[Adsorption, separation, and purification of cyclosporine A using reversed-phase liquid chromatography].

High performance liquid chromatography (HPLC) is widely used in the separation and analysis of cyclosporine A (CsA). Analyzing the chromatographic behavior of CsA is key to the purification of CsA by preparative HPLC. In this study, the retention behavior of CsA on the C18 column using mobile phases of methanol-water and acetonitrile-water was compared. The retention time of CsA was sensitive to the change in the ratio of the organic solvent. When 84%-88% methanol or 75%-85% acetonitrile was used, the retention factor (k) was in the range of 3-7. The change in the peak shape of CsA was investigated with loading amounts of 5, 25, 50, 100, and 500 mg. With an increase in sample loading, the peak shape of CsA in both mobile phases changed from symmetric to tailing, and the retention time reduced. Therefore, it is necessary to focus on the removal of impurities that were eluted before CsA during the purification. In addition, the peak shapes of CsA in methanol-water and acetonitrile-water were similar in the tested concentration range. This indicates that it was not possible to tune the peak shape of CsA by changing the organic solvent. Adsorption isotherms were obtained to describe the retention behavior of CsA. When the mass concentration of CsA in the mobile phase was low, the effect of the organic solvent ratio on the adsorption capacity of CsA on the C18 stationary phase was not distinct. With an increase in the solute mass concentration above 0.5 g/L, the reduced proportion of organic solvent helped improve the adsorption capacity of CsA. When the mass concentration of CsA in the mobile phase reached 5 g/L, the adsorption capacities were 24.9 g/L in 88% methanol and 40.8 g/L in 84% methanol. The adsorption capacity of CsA in acetonitrile-water was higher than that in methanol-water. When the mass concentration of CsA was 5 g/L, the adsorption capacity increased to 46.4 g/L in 75% acetonitrile. Scatchard analysis showed that the slope of the adsorption isotherm decreased gradually, which was consistent with trend observed in the Langmuir adsorption isotherm for the shape of the Langmuir peak (i.e. trailing peak). When the mass concentration of CsA in the mobile phase was between 0.01 g/L and 0.03 g/L, the slope of the curve decreased significantly, and the peak shape of CsA rapidly tailed with increasing loading amount. However, when using a mobile phase with a lower proportion of organic solvent (84% methanol or 75% acetonitrile), this trend was weakened. The adsorption data of CsA were fitted to models. The Langmuir model was found to be suitable for the methanol-water mobile phase, and the Moreau model for the acetonitrile-water mobile phase. The model parameters indicated that the monolayer adsorption of CsA occurred on the C18 stationary phase in both mobile phases, the difference being that more intermolecular interactions between CsA occurred in the acetonitrile-water mobile phase, resulting in a higher adsorption capacity. In methanol-water, the intermolecular interactions between CsA were inhibited by methanol due to its role as a proton donor. As an aprotic solvent, acetonitrile could only weakly inhibit these interactions; hence, the interactions could be improved by increasing the acetonitrile proportion. As the proportion of acetonitrile changed from 85% to 75%, the saturated adsorption capacity increased from 123 g/L to 197 g/L, while the interaction constant decreased from 0.618 to 0.588. Finally, CsA was purified using the conditions of 0-60 min 65%-75% acetonitrile, 60-80 min 75% acetonitrile, by which the impurity could be controlled to below 0.2%. The results of this study will aid in the purification of CsA by preparative HPLC.

Isolation of achiral aliphatic acid derivatives from Piper kadsura using preparative two-dimensional chiral supercritical fluid chromatography.

The separation of structural analogues in natural products has always been one of the challenges in separation science, where supercritical fluid chromatography (SFC) with chiral stationary phases (CSPs) is an unconventional but potential solution. In this study, a preparative two-dimensional chiral SFC (2D cSFC) method that was established with two kinds of CSPs was applied in the isolation of the aliphatic acid derivatives in Piper kadsura (P. kadsura). The RPLC unseparated peaks of two samples A and B of P. kadsura were evenly scattered on the CSP-1 column while they clustered into two groups on the CSP-2 column by SFC. There was impressively complementary selectivity between CSP-1 and CSP-2, which were used for construction of 2D cSFC. The first dimension (1D) separation with CSP-1 fractionated the sample A into six parts by a heart-cutting method and the sample B into nine parts for a comprehensive 2D analysis; then 29 and 71 peaks were respectively found in these parts in the second dimension (2D) separation with CSP-2. Further through 2D preparative separation, 19 high purity components were obtained, and the chemical structures of two of them were confirmed, including a novel unsaturated aliphatic acid compound (8Z,10Z)-12-methoxyheptadeca-8,10-dienoic acid and a known octadecadienoic acid lactone Lactariolide. The 2D cSFC method presented the superiority of separating the achiral compounds of complex samples.

Separation and characterization of phenylamides from Piper kadsura using preparative supercritical fluid chromatography and ultra-high-performance supercritical fluid chromatography-tandem mass spectrometry.

A preparative supercritical fluid chromatography method for the separation of Piper kadsura obtained five phenylamide compounds, which had the same structural skeleton, but changed in the number and position of methoxyl substituents. To improve the separation selectivity of these structural analogues, silica, phenyl, and chiral stationary phases were screened. Only through the combination of Chiral C and phenyl columns could the separation of the five phenylamides be solved. The two-step strategy using preparative supercritical fluid chromatography presented good orthogonality that ensured the purity of the phenylamides. Then, an ultra-high-performance supercritical fluid chromatography hyphened tandem mass spectrometry method was developed, and the fragmentation pattern of phenylamides was summarized. It mainly cleaved in the amide bond to produce the fragment ion, which could help to judge the substituent positions. Twenty-eight possible molecular weights of hydroxyl and methoxyl substituted phenylamides were calculated and screened. Nine compounds were extracted in three [M + H]+ ions at m/z 284.13, 314.13, and 344.13, including five purified compounds and the other four positional or trans-cis phenylamide isomers in low content. The methods developed in this research were useful in the separation and characterization of phenylamide analogues.

A ternary eluent strategy to tune the peak shape of steviol glycosides in reversed-phase liquid chromatography.

In this paper, an effective strategy of using acetonitrile-methanol-water as mobile phase was developed to achieve acceptable peak shape of steviol glycosides in reversed-phase liquid chromatography (RPLC). The change of elution profiles of rebaudioside A (RA) was systematically investigated. Two classical distributions, namely, tailing and fronting peaks resulting from injections of RA solution in range of 0.5-5 mg were both observed in a ternary eluent of acetonitrile-methanol-water (21:43:36, v/v). Next, a three-phase diagram of tailing factor (Tf) was illustrated, showing high dependence of elution profile of RA on the ternary composition. The peak shape of RA can be adjusted mainly based on the additive effect, that is, acetonitrile is more strongly adsorbed to the stationary phase than RA in the pure weak solvent (H2O). Therefore, with the increase of acetonitrile concentration in the ternary eluent, the RA band profiles went from being tailing to fronting shapes. At the same time, due to the large RA-RA interactions, there was anti-Langmuir adsorption isotherm in acetonitrile-water mobile phase, which is the reason for the fronting peaks of RA. It could be concluded that the way of using the ternary eluent of acetonitrile-methanol-water does control and tune the peak shape of steviol glycosides in RPLC separation.

Bioactivity-guided separation of antifungal compounds by preparative high-performance liquid chromatography.

Bioactivity-guided chromatographic methods are of great significance for the isolation of the active compounds in complex samples. In this study, four anti-fungal compounds were located by activity screening and successfully isolated from a microbial fermentation sample by preparative high-performance liquid chromatography. Separation performance of columns including C18, positively charged C18, negatively charged C18 and C8 were firstly investigated. And it showed a better capacity of mixed-mode stationary phases for retention and separation. Therefore, the positively charged C18 column was used to separate the sample into several fractions, among which the active one was identified by the antifungal test. And then the active fraction was enriched and separated again by successively using the negatively charged C18 and C8 columns to obtain four compounds, which were identified as polyoxins A, K, F and H. With activity verification, four polyoxins were found to have good inhibitory effects against the three fungal plant diseases including rice sheath blight, tomato grey mould disease, and apple spot leaf disease.

Isolation of three polyoxins by reversed-phase liquid chromatography with pure aqueous mobile phase.

Developing methods for the isolation of highly polar compounds from complex samples is of great significance. In this study, three polyoxins were successfully isolated from a complex sample (PN1-1# ) by preparative high-performance liquid chromatography. Separation was carried out on five polar reversed-phase stationary phases, using pure aqueous as mobile phase, where the C18HC column can provide the best performance for PN1-1# . Next, the effects of the mobile phase composition were studied. It was found that adding NaClO4 can enhance the retention and resolution, and adding NaH2 PO4 was beneficial to maintain good peak shapes when the sample loading increased. Therefore, the optimized mobile phase consisting of 20 mmol NaH2 PO4 and 20 mmol NaClO4 (adding H3 PO4 to adjust pH 2) was used to separate PN1-1# . This method of using 100% aqueous phase can effectively improve both the retention and the solubility of polar samples. Eventually, through further purification, three compounds, namely, polyoxins B, D, and G, were obtained. This paper provided an effective and eco-friendly strategy for the preparative-scale separation of polar samples.

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.

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%.