Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography: Orthogonality in separation of Polygonum cuspidatum Sieb. et Zucc

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography was investigated in separation of crude ethanol extract from traditional Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. Two‐dimensional contour plots for countercurrent chromatography with high‐performance liquid chromatography was obtained after comprehensive separation was completed. Total peak capacity was evaluated and approximately 810 peaks were obtained through a comprehensive two‐dimensional separation. A highly orthogonality of 52.23% and a large separation space occupancy of 88.86% were achieved. Meanwhile, it was found that several components could be well separated by countercurrent chromatography while they could not be separated by high‐performance liquid chromatography, and vice versa, which further indicated the orthogonality of the two separation methods. The off‐line comprehensive two‐dimensional countercurrent chromatography with high‐performance liquid chromatography provided a promising and powerful method for separation of complex natural products.     

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.     

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.     

Preparative separation of isoquinoline alkaloids from Corydalis impatiens using a middle‐pressure chromatogram isolated gel column coupled with two‐dimensional liquid chromatography

We established a two‐dimensional strong cation exchange/reversed‐phase liquid chromatography protocol to isolate and purify isoquinoline alkaloids from Corydalis impatiens. Isoquinoline alkaloids were first enriched from a C. impatiens extract in which liposoluble components were removed using a medium‐pressure chromatographic tower containing middle chromatogram isolated gel. A strong cation exchange column was employed to separate and obtain 30 fractions. We chose fractions 22–29 for reversed‐phase liquid chromatography purification using characteristic isoquinoline alkaloid ultraviolet absorption spectra. Several isoquinoline alkaloid fractions (22–29) were further separated, and those of low resolution were isolated via two‐dimensional liquid chromatography in the orthogonal plane. A total of eight novel isoquinoline alkaloids with characteristic ultraviolet spectra were obtained from C. impatiens. We thus demonstrate the benefits of off‐line two‐dimensional strong cation exchange/reversed‐phase liquid chromatography to isolate isoquinoline alkaloids from C. impatiens.     

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.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

Online high‐pH reversed‐phase liquid chromatography × low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension for the analysis of alkaloids in Macleaya cordata (willd.) R. Br

An online high‐pH reversed‐phase liquid chromatography× low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension was constructed to separate and identify alkaloids from Macleaya cordata (willd.) R. Br. The modulation was performed by using a dual second dimensional columns interface combined with a make‐up dilution pump, which is responsible for dilution and neutralization of the first dimensional effluent, and the dual second dimensional columns integrated the trapping and the separation function to reduce the second dimension system dead volume. Taking advantage of the dissociable characteristics of alkaloids, mobile phases with different pH values were applied in the first dimension (pH 9.0) and the second dimension (pH 2.6) to improve the orthogonality of two‐dimension separation. Besides, the pulse elution gradient in first dimension and second dimensional gradient were carefully optimized and much better separation was achieved compared to the separation with the traditional two‐dimensional liquid chromatography approach. Finally, mass measurement was performed for alkaloids in M. cordata (willd.) R. Br. by coupling proposed two‐dimensional liquid chromatography system with triple quadrupole mass spectrometry, and 39 alkaloids were successfully identified by comparing the obtained result with the former reported results.     

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.     

Separation of five compounds from leaves of Andrographis paniculata (Burm. f.) Nees by off‐line two‐dimensional high‐speed counter‐current chromatography combined with gradient and recycling elution

An off‐line two‐dimensional high‐speed counter‐current chromatography method combined with gradient and recycling elution mode was established to isolate terpenoids and flavones from the leaves of Andrographis paniculata (Burm. f.) Nees. By using the solvent systems composed of n‐hexane/ethyl acetate/methanol/water with different volume ratios, five compounds including roseooside, 5,4′‐dihydroxyflavonoid‐7‐O‐β‐d ‐pyranglucuronatebutylester, 7,8‐dimethoxy‐2′‐hydroxy‐5‐O‐β‐d ‐glucopyranosyloxyflavon, 14‐deoxyandrographiside, and andrographolide were successfully isolated. Purities of these isolated compounds were all over 95% as determined by high‐performance liquid chromatography. Their structures were identified by UV, mass spectrometry, and ¹H NMR spectroscopy. It has been demonstrated that the combination of off‐line two‐dimensional high‐speed counter‐current chromatography with different elution modes is an efficient technique to isolate compounds from complex natural product extracts.     

An efficient strategy for the extraction and purification of lignans from Schisandra chinensis by a combination of supercritical fluid extraction and high‐speed counter‐current chromatography

An efficient strategy for extracting and separating five lignans from Schisandra chinensis (Turcz.) Baill has been developed using supercritical fluid extraction (SFE) and high‐speed counter‐current chromatography (HSCCC) in the present study. First, the extraction was performed by a preparative SFE system under 15 MPa of pressure at 36°C for 4 h. Then, the SFE extract was successfully separated and purified by HSCCC with a two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water (6:4:5:5, 6:4:6:4, 6:4:8:2, v/v) in a stepwise elution mode. The fractions were analyzed by HPLC, and the chemical structures of the products were identified by ESI‐MS and ¹H NMR spectroscopy. As a result, a total of 12.5 mg of schisandrin at 98.0% purity, 7.1 mg of gomisin A at 98.1% purity, 1.8 mg of schisantherin B at 93.3% purity, 4.4 mg of deoxyschisandrin at 92.9% purity, and 6.8 mg of γ‐schisandrin at 89.1% purity were obtained from 300 mg crude extract in a one‐step purification.     

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.     

Separation and purification of two taxanes and one xylosyl‐containing taxane from Taxus wallichiana Zucc.: A comparison between high‐speed countercurrent chromatography and reversed‐phase flash chromatography

10‐Deacetylbaccatin III, an important semisynthetic precursor of paclitaxel and docetaxel, can be extracted from Taxus wallichiana Zucc. A process for the isolation and purification of 10‐deacetylbaccatin III ( 1 ), baccatin III ( 2 ), and 7β‐xylosyl‐10‐deacetyltaxol ( 3 ) from the leaves and branches of Taxus wallichiana Zucc. via macroporous resin column chromatography combined with high‐speed countercurrent chromatography or reversed‐phase flash chromatography was developed in this study. After fractionation by macroporous resin column chromatography, 80% methanol fraction was selected based on high‐performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis. A solvent system composed of n‐hexane, ethyl acetate, methanol, and water (1.6:2.5:1.6:2.5, v/v/v/v) was used for the high‐speed countercurrent chromatography separation at a flow rate of 2.5 mL/min. The reversed‐phase flash chromatography separation was performed using methanol/water as the mobile phase at a flow rate of 3 mL/min. The high‐speed countercurrent chromatography separation produced compounds 1 (10.2 mg, 94.4%), 2 (2.1 mg, 98.0%), and 3 (4.6 mg, 98.8%) from 100 mg of sample within 110 min, while the reversed‐phase flash chromatography separation purified compounds 1 (9.8 mg, 95.6%) and 3 (4.9 mg, 97.9%) from 100 mg of sample within 120 min.     

Separation of epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography using hydroxypropyl‐β‐cyclodextrin as an additive

The separation of ten epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as the mobile phase additive was investigated, and methods for the analysis of all the epimeric esters by reversed‐phase high‐performance liquid chromatography were established. A biphasic solvent system composed of n‐hexane/20–70% methanol containing 50 mmol/L of hydroxypropyl‐β‐cyclodextrin (1:1, v/v) was selected, which provided high separation factors for five of the epimeric esters, and successful separations by countercurrent chromatography were achieved. The complete separation of five pairs of epimeric ester was obtained with the purity being over 98% for each peak fractions, as determined by high‐performance liquid chromatography. The recovery of each analyte from the eluted fractions reached around 80–88%.     

Separation and purification of four compounds from Desmodium styracifolium using off‐line two‐dimensional high‐speed counter‐current chromatography

An off‐line 2D high‐speed counter‐current chromatography technique in preparative scale has been successfully applied to separate and purify the main compounds from the ethyl acetate extract of Desmodium styracifolium. A two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water at an optimized volume ratio of 1:2:1:2 v/v/v/v was used. Conventional high‐speed counter‐current chromatography was used as the first dimension, and the upper phase of the solvent system was used as the stationary phase in the head‐to‐tail elution mode at a flow rate of 2.0 mL/min and a rotation speed of 900 rpm. Recycling high‐speed counter‐current chromatography served as the second dimension to separate an impure fraction of the first dimension. A total of four well‐separated substances including vanillic acid ( 1 ), β‐sitosterol ( 2 ), formononetin ( 3 ), and aromadendrin ( 4 ) were obtained, and their purities and structures were identified by HPLC–MS and ¹H NMR spectroscopy. The results illustrated that off‐line 2D high‐speed counter‐current chromatography is an effective way to isolate compounds in complex samples.     

Towards eco‐friendly secondary plant metabolite quantitation: Ultra high performance supercritical fluid chromatography applied to common vervain (Verbena officinalis L.)

This report presents the first ultra high performance supercritical fluid chromatography diode array detector based assay for simultaneous determination of iridoid glucosides, flavonoid glucuronides, and phenylpropanoid glycosides in Verbena officinalis (Verbenaceae) extracts. Separation of the key metabolites was achieved in less than 7 min on an Acquity UPC² Torus Diol column using a mobile phase gradient comprising subcritical carbon dioxide and methanol with 0.15% phosphoric acid. Method validation for seven selected marker compounds (hastatoside, verbenalin, apigenin‐7‐O‐glucuronide, luteolin‐7‐O‐glucuronide, apigenin‐7‐O‐diglucuronide, verbascoside, and luteolin‐7‐O‐diglucuronide) confirmed the assay to be sensitive, linear, precise, and accurate. Head‐to‐head comparison to an ultra high performance liquid chromatography comparator assay did prove the high orthogonality of the methods. Quantitative result equivalence was evaluated by Passing‐Bablok‐correlation and Bland‐Altman‐plot analysis. This cross‐validation revealed, that one of the investigated marker compound peaks was contaminated in the ultra high performance liquid chromatography assay by a structurally related congener. Taken together, it was proven that the ultra high performance supercritical fluid chromatography instrument setup with its orthogonal selectivity is a true alternative to conventional reversed phase liquid chromatography in quantitative secondary metabolite analysis. For regulatory purposes, assay cross‐validation with highly orthogonal methods seems a viable approach to avoid analyte overestimation due to coeluting, analytically indistinguishable contaminants.     

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.     

High‐performance liquid chromatographic and subcritical fluid chromatographic separation of α‐arylated ß‐carboline,N‐alkylated tetrahydroisoquinolines and their bioisosteres on polysaccharide‐based chiral stationary phases

New, pharmacologically interesting chiral amino compounds, namely, stereoisomers of α‐hydroxynaphthyl‐ß‐carboline, benz[d]azepine and benz[c]azepine analogs as well as N‐α‐hydroxynaphthylbenzyl‐substituted isoquinolines were enantioseparated by high‐performance liquid chromatographic and subcritical fluid chromatographic methods on polysaccharide‐based chiral stationary phases. Separation of the stereoisomers was optimized in both subcritical fluid chromatography and normal phase liquid chromatographic modes by investigating the effects of the composition of the bulk solvent, temperature, and the structures of the analytes and selectors. Both normal phase liquid chromatography and subcritical fluid chromatography exhibited satisfactory performance, albeit with somewhat different effectiveness in the separation of the stereoisomers studied. The optimized methods offer the possibility to apply preparative‐scale separations thereby enabling further pharmacological investigations of the enantiomers.     

Residue measurement of pendimethalin in tobacco by using heart‐cutting two dimensional liquid chromatography coupled with tandem mass spectrometry

A novel heart‐cutting two‐dimensional liquid chromatography coupled with tandem mass spectrometry method was developed for quantitative analysis of pendimethalin residue in tobacco. The strategy of reversed phase liquid chromatography coupled with another reversed‐phase liquid chromatography was employed for high column efficiency and excellent compatibility of mobile phase. In the first dimensional chromatography, a cyano column with methanol/water as the eluent was applied to separate pendimethalin from thousands of interference components in tobacco. By heart‐cutting technique, which effectively removed interference components, the target compound was cut to the second dimensional C18 column for further separation. The pendimethalin residue was finally determined by the tandem mass spectrometry under multiple reaction monitoring reversed‐phase liquid chromatography mode. Sample pretreatment of the new method was simplified, involving only extraction and filtration. Compared with traditional methodologies, the new method showed fairly high selectivity and sensitivity with almost no matrix interference. The limit of quantitation for pendimethalin was 1.21 ng/mL, whereas the overall recoveries ranged from 95.7 to 103.3%. The new method has been successfully applied to non‐stop measure of 200 real samples, without contamination of ion source. Detection results of the samples agreed well with standard method.     

Synthesis and evaluation of a maltose‐bonded silica gel stationary phase for hydrophilic interaction chromatography and its application in Ginkgo Biloba extract separation in two‐dimensional systems

Maltose covalently bonded to silica was prepared by using carbonyl diimidazole as a cross‐linker and employed as a stationary phase for hydrophilic interaction liquid chromatography. The column efficiency and the effect of water content, buffer concentration, and pH value influenced on retention were investigated. The separation or enrichment selectivity was also studied with nucleosides, saccharides, amino acids, peptides, and glycopeptides. The results indicated that the stationary phase processed good separation efficiency and separation selectivity in hydrophilic interaction liquid chromatography mode. Moreover, a two‐dimensional hydrophilic interaction liquid chromatography× reversed‐phase liquid chromatography method with high orthogonality was developed to analyze the Ginkgo Biloba extract fractions. The development of this two‐dimensional chromatographic system would be an effective tool for the separation of complex samples of different polarities and contents.     

Development of an automated dual‐mode supercritical fluid chromatography and reversed‐phase liquid chromatography mass‐directed purification system for small‐molecule drug discovery

We report the development of a dual‐mode mass‐directed supercritical fluid chromatography and reversed‐phase liquid chromatography purification system. The addition of a third pump allows for flexible mobile phase control between the two techniques, and enables operation of either chromatography mode within minutes by activation of a set of switching valves on a single system. Software control, fluidic pathways, interface to the mass spectrometer, and fraction collection have been modified for compatibility between both separation methods. The conditioning solvent and tuning parameters for the mass spectrometer were adjusted to achieve an ideal signal trace in either mode with good linearity (r² > 0.970) over a range of concentrations and minimal noise for accurate peak detection and isolation. The registration success rate is 90% and overall sample recovery for either technique is 80?90%. Combining two orthogonal separation and purification modes in one single system has improved the purification throughput of complex mixtures and has been a valuable, cost‐saving tool in our laboratory.