Ian Garrard

Dibenzothiophene (DBT) is a typical recalcitrant thiophenic sulfur component of fuels, and its desulphurization has been a model reaction in the treatment of these compounds. Based on this information, the potential of Pseudomonas fluorescens (UCP 1514) on the desulfurization of dibenzothiphene was studied, in order to use it for reducing the sulfur content of diesel oil in compliance with environmental regulations. The result of biodegradation by the bacteria was determined by undertaking high-performance liquid chromatography of the metabolites produced. These can also be identified by gas chromatography with a mass spectrometry detector, and doing so revealed a sulfur-free product, biphenyl, as the final product of the degradation process. The results showed a decrease of 73% in dibenzothiophene content, which means that P. fluorescens removes sulfur from dibenzothiophene with a good selectivity to form biphenyl. These promising results indicate that P. fluorescens has an interesting potential to degrade sulfur-containing compounds in diesel oil and thereby could help in removing sulfur content from diesel oil. The process of microbial desulfurization described herein can be used particularly after carrying out hydrodesulfurization. Consequently, the sulfur content could be reduced even further. Applying P. fluorescens UCP 1514 in dibenzothiophene could help to understand the nature of the biodegradation process and to achieve the regulatory standards for sulfur level in fossil fuels.

This review is a comprehensive classification of solvent systems used for natural product purifications in countercurrent and centrifugal partition chromatography.

High performance countercurrent chromatography (HPCCC) was successfully applied for the separation of nostotrebin 6 from cultivated soil cyanobacteria in a two-step operation. A two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (4:5:4:5, v/v/v/v) was employed for the HPCCC separation. In the first-step operation, its neutral upper phase was used as stationary phase and its basic lower phase (1% NH₃ in lower phase) was employed as mobile phase at a flow rate of 1 mL/min. In the second operation step, its neutral upper phase was used as stationary phase, whereas both its neutral lower phase and basic lower phase were employed as mobile phase with a linear gradient elution at a flow rate of 0.8 mL/min. The revolution speed and temperature of the separation column were 1,000 rpm and 30 °C, respectively. Using HPCCC followed by clean-up on Sephadex LH-20 gel, 4 mg of nostotrebin 6 with a purity of 99% as determined by HPLC/DAD-ESI-HRMS was obtained from 100 mg of crude extract. The chemical identity of the isolated compound was confirmed by comparing its spectroscopic data (UV, ESI-HRMS, ESI-HRMS²) with those of an authentic standard and data available in the literature.

A high‐performance counter‐current chromatography (HPCCC) method was applied for the first time for the preparative separation and purification of three rare compounds which occur as minor constituents in the fruits of Peucedanum alsaticumL.: 5‐substituted coumarin notoptol and two dihydropyranochromones: divaricatol and ledebouriellol. A scale‐up process from analytical to preparative in a very short time was developed. In order to purify a range of rare and minor compounds with different polarity two separate experiments were performed, one in reverse phase, the other in normal phase, using the same crude extract. A two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water (1:1:1:1) was developed. The components purified and collected were analyzed by high‐performance liquid chromatography. The method yielded 0.7 mg of notoptol, 1.46 mg of ledebouriellol at purity of 99.5%, and 10 mg of mixtures of divaricatol, alsaticol and alsaticocoumarin, where divaricatol present 22% by peak area. These amounts were obtained from 1 g of the crude extract in a single run. This is the first time when minor notoptol, ledebouriellol, and divaricatol were isolated in a single run using HPCCC method and first time when these were identified in plant from Peucedanum genus.

The development of the planetary‐motion countercurrent chromatography machine into a high speed, high loading and high resolution dynamic extraction centrifuge has allowed the scale‐up of the technique from analytical to pilot (kg/day). At this scale, solvent usage is a major consideration for both economical and environmental (“green”) reasons. The analysis by gas chromatography of the composition of the two phases in solvent systems allows each layer to be made separately as required, minimising solvent wastage. Furthermore, as operation is generally isocratic, analysis of the recovered condensate from evaporated fractions allows the mobile phase to be easily reconstituted ready for re‐use in the centrifuge. Such recycling further minimises solvent usage during preparative and pilot‐scale separations.