Exploration and optimization of conditions for quantitative analysis of lignans in Schisandra chinensis by an online supercritical fluid extraction with supercritical fluid chromatography system.

An online supercritical fluid extraction with supercritical fluid chromatography system could provide sequential extraction and quantitative analysis of lignans in Schisandra chinensis. Supercritical fluid extraction conditions were optimized at 15 MPa, 50°C, and 4 min with supercritical CO2 adding 1% methanol; the elution volume and flow rate were set at 6 mL and 2 mL/min to blow extract out of the tank completely. The split-flow rate was confirmed at 2.5%, which determines injection volume and accuracy of quantitative detection. The factors having negative influences on supercritical fluid chromatography retention in the online system, including sample loading forms and backpressure settings, are discussed in the paper. At last, an extraction-quantitative method for lignans in Schisandra chinensis was developed, which could be finished within 19.5 min. The total content percentage of four lignans (Schisandrin, Schisandrin A, Schisandrin B and Schisandrol B) in four batches was respectively measured to be 1.42, 1.54, 1.62, and 1.90%.

Design, synthesis and evaluation of a series of alkylsiloxane-bonded stationary phases for expanded supercritical fluid chromatography separations.

Supercritical fluid chromatography (SFC) today represents an alternative technique in analytical chemistry due to its obvious benefits in kinetic performance and its complementarity to liquid chromatography. In this paper, a series of alkylsiloxane-bonded stationary phases were synthesized and evaluated to expand their SFC applications. Five kinds of non-endcapped C8 stationary phases (C8-1 to C8-5) with increasing bonding density were synthesized, and the carbon content was 3.91%, 6.07%, 7.97%, 8.65% and 9.10% respectively. Retention mechanism of the C8 phases in SFC in SFC was investigated by the use of a linear solvation energy relationship (LSER) model. Results underlined a close relationship between the bonding density of alkyl chain and the dispersion and polar interactions of the stationary phase. Complementary evaluation was studied based on the calculation of vector angle (θ), and the widest θ of 123° was found between silica and C8 with the highest bonding density. Selective diversity also existed between the two C8 phases with the highest and lowest bonding densities. In addition, the effect of modifier on the SFC mechanism was investigated. Modifiers (methanol, ethanol, isopropanol and acetonitrile) had insignificant influence on the dispersion interaction but they mainly affected the hydrogen bonding interaction by changing the LSER parameters a and b. Finally, C8 and silica columns were applied for separation of eight amide alkaloids of Piper kadsura. Silica provided better retention but limited selectivity while C8 can distinguish alkaloids different in alkyl chain, double bond and cis-trans structure. This research further contributed to demonstrate the potential of alkylsiloxane-bonded stationary phase in improving selectivity of SFC.

[Separation and purification of compounds from piper kadsura using preparative reversed-phase liquid chromatography and preparative supercritical fluid chromatography].

A method based on preparative reversed-phase liquid chromatography (prep-RPLC) and preparative supercritical fluid chromatography (prep-SFC) was developed for the separation and purification of compounds from piper kadsura. A pretreatment method was first developed, including methanol extraction, water precipitation, petroleum ether extraction, etc. Chlorophyll and other strong polar impurities were removed from the piper kadsura samples, and the target components were enriched in petroleum ether extracts. The piper kadsura samples were separated into 18 fractions on a Unitary C18 column (250 mm×20 mm, 5 µm) with water and methanol as the mobile phases. Then, the SFC parameters, including the column, modifier, temperature, and backpressure were optimized. The optimized conditions for prep-SFC were as follows:XAmide column (250 mm×20 mm, 5 µm), methanol as the modifier, 30℃ column temperature, and 15.0 MPa backpressure. Because of the good orthogonality of RPLC and SFC, six highly pure compounds were isolated, including kadsurenone, wallichinine, denudatin B, pellitorine, 2E-decenoic acid N-isobutylamide, and futoxide.

[Separation and purification of the components in Trachelospermum jasminoides by two dimensional hydrophilic interaction liquid chromatography- reversed-phase liquid chromatography].

A preparative two dimensional hydrophilic interaction liquid chromatography/reversed-phase liquid chromatography (Pre-2D-HILIC/RPLC) method was established to separate and purify the components in Trachelospermum jasminoides. The pigments and strongly polar components were removed from the crude extract after the active carbon decolorization and solid phase extraction processes. A Click XIon column (250 mm×20 mm, 10 µm) was selected as stationary phase and water-acetonitrile as mobile phases in the first dimensional HILIC. Finally, 15 fractions were collected under UV-triggered mode. In the second dimensional RPLC, a C18 column (250 mm×20 mm, 5 µm) was selected and water-acetonitrile was used as mobile phases. As a result, 14 compounds with high purity were obtained, which were further identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Finally, 11 lignan compounds and three flavonoid compounds were obtained. The method has a good orthogonality, and can improve the resolution and the peak capacity. It is significant for the separation of complex components from Trachelospermum jasminoides.

[Application of off-line two dimensional reversed-phase liquid chromatography/supercritical fluid chromatography to the separation of the seeds of Trichosanthes kirilowii Maxim].

An off-line two dimensional reversed-phase liquid chromatography/supercritical fluid chromatography (2D RPLC/SFC) method was developed for the separation of the seeds of Trichosanthes kirilowii Maxim. (T. kirilowii.). In the experiment, RPLC was used in the first dimension to analyze the samples. And 12 fractions (F1-F12) were obtained according to the chromatographic peak collection mode, the fractions were re-analyzed in both RPLC and SFC modes. The results clearly showed that this 2D method had good orthogonality. The SFC was used as the second dimension. Ethanol-n-hexane (3:7, v/v) was used as a modifier in SFC. The modifier could provide appropriate elution power. When the sample load increased, it could also ensure the solubility of samples. The 2D RPLC/SFC method had obvious advantages for separating the compounds with similar properties, enriching and purifying the trace components. Finally, 150 peaks could be detected. The off-line 2D RPLC/SFC method will be further enlarged to a preparative level for preparation of compounds. The method can provide a practical reference for the purification of chemical constituents and material basis research of the seeds of T. kirilowii. in the future.

Preparative separation of the polar part from the rhizomes of Anemarrhena asphodeloides using a hydrophilic C18 stationary phase.

The goal of this study was to develop a method that utilized a hydrophilic C18 stationary phase in the preparative high performance liquid chromatography to isolate the polar part from the rhizomes of Anemarrhena asphodeloides. The results showed that an initial mobile phase of pure water for the separation could greatly increase the retention and solubility of the polar compounds at the preparative scale. Introducing polar groups on the surface of the hydrophilic C18 column together with the use of optimized mobile phase compositions improved the column separation selectivity for polar compounds. Eleven previously undescribed compounds in Anemarrhena asphodeloides were obtained, indicating that the method developed in this study would facilitate the purification and separation of the polar part of traditional Chinese medicines.

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.

Rapid purification of diastereoisomers from Piper kadsura using supercritical fluid chromatography with chiral stationary phases.

Supercritical fluid chromatography (SFC) with chiral stationary phases (CSPs) is an advanced solution for the separation of achiral compounds in Piper kadsura. Analogues and stereoisomers are abundant in natural products, but there are obstacles in separation using conventional method. In this paper, four lignan diastereoisomers, (-)-Galbelgin, (-)-Ganschisandrin, Galgravin and (-)-Veraguensin, from Piper kadsura were separated and purified by chiral SFC. Purification strategy was designed, considering of the compound enrichment, sample purity and purification throughput. Two-step achiral purification method on chiral preparative columns with stacked automated injections was developed. Unconventional mobile phase modifier dichloromethane (DCM) was applied to improve the sample solubility. Four diastereoisomers was prepared at the respective weight of 103.1mg, 10.0mg, 152.3mg and 178.6mg from 710mg extract with the purity of greater than 98%.

Development, validation and application of a hydrophilic interaction liquid chromatography-evaporative light scattering detection based method for process control of hydrolysis of xylans obtained from different agricultural wastes.

Purified standards of xylooligosaccharides (XOSs) (DP2-6) were first prepared from a mixture of XOSs using solid phase extraction (SPE), followed by semi-preparative liquid chromatography both under hydrophilic interaction liquid chromatography (HILIC) modes. Then, an accurate quantitative analysis method based on hydrophilic interaction liquid chromatography-evaporative light scattering detection (HILIC-ELSD) was developed and validated for simultaneous determination of xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6). This developed HILIC-ELSD method was applied to the comparison of different hydrolysis methods for xylans and assessment of XOSs contents from different agricultural wastes. The result indicated that enzymatic hydrolysis was preferable with fewer by-products and high XOSs yield. The XOSs yield (48.40%) from sugarcane bagasse xylan was the highest, showing conversions of 11.21g X2, 12.75g X3, 4.54g X4, 13.31g X5, and 6.78g X6 from 100g xylan.

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.

Hydrophilic interaction liquid chromatography for the separation, purification, and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz.

A systematic strategy based on hydrophilic interaction liquid chromatography was developed for the separation, purification and quantification of raffinose family oligosaccharides from Lycopus lucidus Turcz. Methods with enough hydrophilicity and selectivity were utilized to resolve the problems encountered in the separation of oligosaccharides such as low retention, low resolution and poor solubility. The raffinose family oligosaccharides in L. lucidus Turcz. were isolated using solid-phase extraction followed by hydrophilic interaction liquid chromatography at semi-preparative scale to obtain standards of stachyose, verbascose and ajugose. Utilizing the obtained oligosaccharides as standards, a quantitative determination method was developed, validated and applied for the content determination of raffinose family oligosaccharides both in the aerial and root parts of L. lucidus Turcz. There were no oligosaccharides in the aerial parts, while in the root parts, the total content was 686.5 mg/g with the average distribution: raffinose 66.5 mg/g, stachyose 289.0 mg/g, verbascose 212.4 mg/g, and ajugose 118.6 mg/g. The result provided the potential of roots of L. lucidus Turcz. as new raffinose family oligosaccharides sources for functional food. Moreover, since the present systematic strategy is efficient, sensitive and robust, separation, purification and quantification of oligosaccharides by hydrophilic interaction liquid chromatography seems to be possible.

Preparation and chromatographic evaluation of a newly designed steviol glycoside modified-silica stationary phase in hydrophilic interaction liquid chromatography and reversed phase liquid chromatography.

A diterpene glycoside compound, rebaudioside A (commonly abbreviated as RA), was immobilized onto porous silica surface through "thiol-ene" click chemistry strategy. The successful immobilization of the RA on the silica support was confirmed by FT-IR and elemental analysis. Chromatographic characteristics of the new stationary phase, named Click TE-RA, were evaluated by a set of diverse analytes such as carbohydrates, nucleosides, and organic acids in hydrophilic interaction liquid chromatography (HILIC) mode. The effects of water content, buffer pH and concentration were investigated and a typical HILIC retention feature of Click TE-RA was observed at high organic modifier content. The Click TE-RA stationary phase was further studied by a series of glycoside compounds. Tunable retention mechanisms from hydrophilic to hydrophobic interactions were observed. Separation of very polar compounds including oligosaccharides, nucleic acid bases and nucleosides using Click TE-RA in HILIC mode was successfully accomplished. In addition, separation of saponins both in HILIC and reversed-phase liquid chromatography (RPLC) modes was performed, demonstrating the presence of orthogonality between two different modes on Click TE-RA column. The multiple interactions induced by polar sugar group and hydrophobic aglycone group allowed this Click TE-RA to serve as a multi-mode stationary phase in two-dimensional liquid chromatography.

Alkaloids analysis using off-line two-dimensional supercritical fluid chromatography × ultra-high performance liquid chromatography.

In this study, an off-line two-dimensional (2-D) supercritical fluid chromatography (SFC) × ultra-high performance liquid chromatography (UHPLC) method with high orthogonality was developed for the analysis of the practical amide alkaloids fraction from P. longum L. The effects of SFC parameters such as column type, organic modifier, temperature and back-pressure on separation were systematically evaluated. Different selectivity was observed for different columns (BEH, BEH 2-EP, XAmide and CSH FP). An investigation was then carried out of the orthogonality of different columns and systems following a geometric approach with a set of amide alkaloid samples. The orthogonality between a CSH FP column and a BEH column reached 50.79%, which was much higher than that for the other columns. While the orthogonality between SFC and UHPLC based on an XAmide column and an HSS T3 column reached 69.84%, which was the highest of all the combinations. At last, the practical amide alkaloids fraction was analyzed with an off-line 2-D chromatography SFC × UHPLC system. In total, at least 340 peaks were detected by this method. Rapid separation in these two dimensions and easy post treatment of SFC facilitated this 2-D system for the separation of complex samples.

[Structural characterization of Astragalus polysaccharides using partial acid hydrolysis-hydrophilic interaction liquid chromatography-mass spectrometry].

Water-soluble polysaccharides from traditional Chinese medicine (TCM) have properties of broad-spectrum treatment and low toxicity, making them as important components in natural medicines and health products. In order to solve the problem of polysaccharides characterization caused by their complex structures, a "bottom-up" approach was developed to complete the characterization of polysaccharides from Astragalus. Firstly, Astragalus pieces were extracted with hot water and then were precipitated by ethanol to obtain Astragalus polysaccharides. Secondly, a partial acid hydrolysis method was carried out and the effects of time, acid concentration and temperature on hydrolysis were investigated. The degree of hydrolysis increased along with the increase of hydrolysis time and acid concentration. The temperature played a great role in the hydrolysis process. No hydrolysis of the polysaccharides occurred at low temperature, while the polysaccharides were almost hydrolyzed to monosaccharide at high temperature. Under the optimum hydrolysis conditions (4 h, 1.5 mol/L trifluoroacetic acid, and 80 °C), Astragalus polysaccharides were hydrolyzed to characteristic oligosaccharide fragments. At last, a hydrophilic liquid chromatography-mass spectrometry method was used for the separation and structural characterization of the polysaccharide hydrolysates. The results showed that the resulting polysaccharides were mainly 1--> 4 linear glucan, and gluco-oligosaccharides with the degrees of polymerization (DP) of 4 - 11 were obtained after partial acid hydrolysis. The significance of this study is that it is the guidance for the characterization of other TCM polysaccharides.