Purification of coenzyme Q10 from fermentation extract: high-speed counter-current chromatography versus silica gel column chromatography

High-speed counter-current chromatography (HSCCC) is applied to the purification of coenzyme Q(10) (CoQ(10)) for the first time. CoQ(10) was obtained from a fermentation broth extract. A non-aqueous two-phase solvent system composed of heptane-acetonitrile-dichloromethane (12:7:3.5, v/v/v) was selected by analytical HSCCC and used for purification of CoQ(10) from 500 mg of the crude extract. The separation yielded 130 mg of CoQ(10) at an HPLC purity of over 99%. The overall results of the present studies show the advantages of HSCCC over an alternative of silica gel chromatography followed by recrystallization. These advantages extend to higher purity (97.8% versus 93.3%), recovery (88% versus 74.3%) and yield (26.4% versus 23.4%). An effort to avoid the toxic, expensive solvent CH(2)Cl(2) was unsuccessful, but at least its percentage is low in the solvent system.     

Requirements for soft magnetic underlayer (SUL)—Micromagnetic simulations of single-pole-type write heads and SUL systems

Landau-Lifshitz-Gilbert micromagnetic models were used to analyze the head field distributions and high-frequency responses for various soft magnetic underlayer thicknesses (t-SUL) and saturation magnetization flux densities (B_s) of single-pole-type (SPT) head-SUL systems. It was found that B_s of 10-12 kG and t-SUL of 30-45 nm would be sufficient for the examined head and perhaps for most next generation head-SUL systems. Antiferromagnetic coupling in the SUL affects the head and SUL magnetization and, eventually, the head field. With regard to the head magnetization response, it was found that the magnetization under the coil responded first, while the response at the main pole tip was quite slow, even compared with the head field. The main pole tip of the head was fully saturated and the main pole yoke was almost saturated, while the SUL was not saturated in the examined head-SUL system.     

Double-modification of lectin using two distinct chemistries for fluorescent ratiometric sensing and imaging saccharides in test tube or in cell

The site-selective incorporation of two different fluorophores into a naturally occurring protein (lectin, a sugar-binding protein) has been successfully carried out using two distinct orthogonal chemical methods. By post-photoaffinity labeling modification, Con A, a glucose- and mannose-selective lectin, was modified with fluorescein in the proximity of the sugar binding site (Tyr100 site), and the controlled acylation reaction provided the site-selective attachment of coumarin at Lys114. In this doubly modified Con A, the fluorescein emission changed upon the binding to the corresponding sugars, such as the glucose or mannose derivatives, whereas the coumarin emission was constant. Thus, the doubly modified Con A fluorescently sensed the glucose- and mannose-rich saccharides in a ratiometric manner while retaining the natural binding selectivity and affinity, regardless of the double modification. On the benefit of the ratiometric fluorescent analysis using two distinct probes, the sugar trimming process of a glycoprotein can be precisely monitored by the engineered Con A. Furthermore, the doubly modified Con A can be used not only for the convenient fluorescent imaging of saccharides localized on a cell surface, such as the MCF-7, a breast cancer cell having rich high-mannose branch, but also for the ratiometric fluorescent sensing of the glucose concentration inside HepG2 cells. These results demonstrated that the semisynthetic lectin modified doubly by two distinct chemistries is superior to the singly modified one in function, and thus, it may be potentially useful in cell, as well as in test tube.     

Addition of chlorodisilanes to 1,3-butadienes in the presence of tetrakis(triphenylphosphine)palladium(0). A facile route to (Z)-1,4-bis(chlorosilyl)but-2-enes

In the presence of a catalytic amount of tetrakis(triphenylphosphine)palladium(0), chlorodisilanes of type Me_nSi₂Cl_6-n (n  0, 2, 3 and 4) add to 1,3-butadienes to give (Z)-1,4-bis(chlorosilyl)but-2-enes.     

The action of tris(triphenylphosphine)chlororhodium on polyhydromonosilanes

Dibromomethyllithium and its homologues RCBr₂Li (R = Bu, Me₃Si, Br) are prepared by metallation of the corresponding α,α-dibromo compounds with lithium diisopropylamide in THF at low temperature. Various coupling reactions are described (alkylation, hydroxyalkylation). New one-step preparations of α-bromoepoxides and α-bromoketones are reported.     

ZnS-Zn nanocables and ZnS nanotubes

ZnS-Zn nanocables and ZnS nanotubes have been synthesized by a thermochemical process in a simple and safe way. The as-prepared nanocables consist of a single crystal Zn core with a diameter of 20 nm and a polycrystalline ZnS sheath with a thickness of 8 nm. The evaporation of the Zn core leads to the formation of ZnS nanotubes.     

SEPARATION OF THE MINOR FLAVONOLS FROM FLOS GOSSYPII BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos Gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v) and (0:1:0:1, v/v). The separation was repeated 3 times, and 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4'-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%) and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, (1)H NMR and (13)C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And four separation rules of flavonols according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC.     

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.     

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.