Separation of tanshinones from Salvia miltiorrhiza bunge by multidimensional counter-current chromatography

Non-equilibrium chromatography (NEC) is a chromatographic mode for the rapid separation of polymers. The retention behavior of various proteins (human, chicken, bovine serum albumin) and supercoiled circular double-stranded DNA (plasmids) was investigated using a phosphate buffer as a mobile phase at different velocities and column temperatures with a C₁ column with very low-packing particle diameter as a stationary phase. It was shown that the two factors (temperature and velocity) constituted important parameters in the retention mechanism of plasmids and proteins in NEC. The protein was retained more than the plasmid. At all the temperatures (5, 10, 15, 20, 25 °C) the plasmid retention increased over the entire flow-rate range (0.02–1.8 ml/min). For the protein, the retention curve presented a decrease in the relative retention time until a critical value of the mobile phase flow-rate, followed by an increase. The transition between the two well known NEC methods, slalom chromatography and hydrodynamic chromatography was clearly visualized for proteins at the lowest temperature, but did not appear for plasmids due to their strong compact structure.     

Well‐Posed Treatment of Space‐Charge Layers in the Electroneutral Limit of Electrodiffusion

The electroneutral model describes cellular electrical activity, accounting for ionic concentration dynamics without resolution of the fine spatial scales of the space‐charge layer. This is done by asserting that the ionic solution is electrically neutral at each point in space. However, electroneutrality is inconsistent with the original boundary conditions at cell membranes. We consider three separate methods of resolving this inconsistency that result in well‐posed models that are accurate approximations to a detailed model in which the space‐charge layer is fully resolved. A particular electrodiffusion problem is utilized to make the discussion specific.© 2015 Wiley Periodicals, Inc.     

Three new nortriterpene glycosides and two new triterpene glycosides from the bulbs of Scilla scilloides

Three new norlanostane-type triterpene glycosides, scillanostasides A, B, and C, and two new lanostane-type triterpene glycosides, scillanostasides D and E, were isolated from the bulbs of Scilla scilloides Druce (Liliaceae) along with one known norlanostane-type triterpene heptaglycoside, scillascilloside G-1. Their chemical structures were determined on the basis of spectroscopic data as well as chemical evidence.     

Stereoselective synthesis of spirocyclic oxindoles based on a one-pot Ullmann coupling/Claisen rearrangement and its application to the synthesis of a hexahydropyrrolo[2,3-b]indole alkaloid

An efficient and convenient approach to the synthesis of spirocyclic oxindoles from iodoindoles has been developed. The most striking feature of this approach is that the sequential intramolecular Ullmann coupling and Claisen rearrangement proceeds in a one-pot manner to afford 3-spiro-2-oxindoles in good yield with excellent diastereoselectivity. Application of this one-pot reaction to chiral non-racemic tertiary alcohol substrates resulted in complete chirality transfer to the spirocyclic quaternary carbon. Using this method, asymmetric total synthesis of (−)-debromoflustramine B was accomplished.     

Rapid preparative separation of six bioactive compounds from Gentiana crassicaulis Duthie ex Burk. using microwave‐assisted extraction coupled with high‐speed counter‐current chromatography

A rapid method combining microwave‐assisted extraction (MAE) and high‐speed counter‐current chromatography (HSCCC) was applied for preparative separation of six bioactive compounds including loganic acid ( I ), isoorientin‐4′‐O‐glucoside ( II ), 6′‐O‐β‐d ‐glucopyranosyl gentiopicroside ( III ), swertiamarin ( IV ), gentiopicroside ( V ), sweroside ( VI ) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. MAE parameters were predicted by central composite design response surface methodology. That is, 5.0 g dried roots of G. crassicaulis were extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by HSCCC with n‐butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail‐to‐head elution mode. 16.3, 8.8, 12., 25.1, 40.7, and 21.8 mg of compounds I–VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPLC with photo diode array detection and quadrupole TOF‐MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, MAE coupled with HSCCC could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.     

pH-zone-refining counter-current chromatography: Origin,mechanism, procedure and applications

Since 1980, high-speed counter-current chromatography (HSCCC) has been used for separation and purification of natural and synthetic products in a standard elution mode. In 1991, a novel elution mode called pH-zone refining CCC was introduced from an incidental discovery that an organic acid in the sample solution formed the sharp peak of an acid analyte. The cause of this sharp peak formation was found to be bromoacetic acid present in the sample solution which formed a sharp trailing border to trap the acidic analyte. Further studies on the separation of DNP-amino acids with three spacer acids in the stationary phase revealed that increased sample size resulted in the formation of fused rectangular peaks, each preserving high purity and zone pH with sharp boundaries. The mechanism of this phenomenon was found to be the formation of a sharp trailing border of an acid (retainer) in the column which moves at a lower rate than that of the mobile phase. In order to facilitate the application of the method, a new method was devised using a set of retainer and eluter to form a sharp retainer rear border which moves through the column at a desired rate regardless of the composition of the two-phase solvent system. This was achieved by adding the retainer in the stationary phase and the eluter in the mobile phase at a given molar ratio. Using this new method the hydrodynamics of pH-zone-refining CCC was diagrammatically illustrated by three acidic samples. In this review paper, typical pH-zone-refining CCC separations were presented, including affinity separations with a ligand and a separation of a racemic mixture using a chiral selector in the stationary phase. Major characteristics of pH-zone-refining CCC over conventional HSCCC are as follows: the sample loading capacity is increased over 10 times; fractions are highly concentrated near saturation level; yield is improved by increasing the sample size; minute charged compounds are concentrated and detected at the peak boundaries; and elution peaks are monitored with a pH flow meter for compounds with no chromophore. Since 1994, over 70 research papers on pH-zone-refining CCC have been published with the trends increasing in the recent years.     

Isolation and characterization of carbene derivatives of La@C82(Cs)

The photochemical reaction of La@C82(Cs) with 2-adamantane-2,3-[3H]-diazirine (1) affords the adduct 2 of La@C82(Cs) with adamantylidene (Ad:) in a high selectivity. The two isomers of La@C82(Cs)(Ad), 2a and 2b, are isolated by HPLC and characterized by electron spin resonance, mass, and UV-vis-near-infrared spectroscopies. The electronic properties of 2a and 2b are very similar to that of the pristine La@C82(Cs), suggesting that 2a and 2b retain the essential electronic and structural character of La@C82(Cs).     

Optical enzyme sensor for determining <Emphasis Type="SmallCaps">l</Emphasis>-lysine content using <Emphasis Type="SmallCaps">l</Emphasis>-lysine oxidase from the rockfish <Emphasis Type="BoldItalic">Sebastes schlegeli</Emphasis>

l-Lysine (l-Lys) in living bodies is critical for metabolism; therefore, determination of its levels in food is important. Most enzymatic methods for l-Lys analysis are performed using l-lysine oxidase (LyOx), but commercially manufactured LyOx is generally not highly selective for l-Lys among amino acids. We previously isolated LyOx as an antibacterial protein secreted from the skin of the rockfish Sebastes schlegeli. In the present study, we developed an optical enzyme sensor system for rapid and continuous determination of l-Lys using this LyOx. The system comprised an immobilized LyOx membrane, an optical oxygen probe, a flow system, and a personal computer. The amount of l-Lys was detected as a decrease in the oxygen concentration due to the LyOx reaction. The specificity of the sensor was examined against various amino acids. The sensor response was specific for l-Lys. Good reproducibility was obtained in 58 assays. The response of the sensor using commercially prepared LyOx was unstable compared with the response using LyOx isolated in our laboratory. Our sensor system could be used for 5 weeks without our having to change the enzyme membrane. The calibration curve for a standard l-Lys solution was linear from 0.1 to 3.0 mmol L⁻¹. One assay could be completed within 2 min. The sensor was applied to determine the l-Lys content in food samples such as bonito cooking water and scallop hepatopancreas. The values obtained using the sensor and conventional high-performance liquid chromatography methods were well correlated.