| A high throughput extraction and preparative separation technology was studied in this thesis. The purpose of this work is to establish small molecule metabolites fraction libraries for preliminary screening of bioactive substances. This work creates the foundation for building a library of active small molecule metabolites from marine microorganisms. More details are as follows:Firstly, to improve the filtration rates, the technology of pretreating fermentation broths of marine bacteria and actinomycetes was studied. With marine bacteria as the main research objects, fermentation broths were pretreated by flocculation. The optimum flocculation was screened by comparing the light-transmittance at610nm, the rates of filtration, the removal ratio of proteins and polysaccharoses from eight kinds of flocculations. A12(SO4)3and poly-AlCl3were chosen as two candidates for further screening. The effects of their application on extracting small molecule metabolites from marine bacteria were investigated by HPLC (high performance liquid chromatography). The effects of four kinds of solid-liquid sepration methods were compared to pretreat the fermentation broths of marine bacteria. The results were as follows:Using A12(SO4)3as flocculant leads to rapid flocculation, high removal ratio of protein and polysaccharose, and pellucid filtration. The application of A12(SO4)3did not increase the Al concent of crude extracts. The optimum flocculation pH is6and the optimum temperature range is25℃to45℃. The dosage of A12(SO4)3and the protein concent of fermentation broths have some correlation. The method is adaptable to most marine bacteria. What is more, the pretreatment method is proved to be adaptive to actinomycetes.Secondly, suitable resins were screened and combined from13different kinds of macroporous resins by comparing extract masses and HPLC fingerprints. The advantages of using three types of resins in group was studied by mass spectrometry. The adsorption and desorption conditions were optimized by monitoring the changing trend of absorbance at254nm and by comparing HPLC fingerprints. Results are as follows:the combination of three types of resins—DM11, HPD400, and SD300, can adsorb more small molecule metabolites from fermentation broths, and to some extent, can optimize the dosage of resins. The suitable collocation ratio of DMll:HPD400:SD300is about2:2:3. The optimal adsorption temperture is5℃and the optimum pH values is5. The suitable flow rate is4BV/h and the the loading amount is about10BV for actinomycetes and32BV for bacteria. The optimal desorption conditions are as follows:the preliminary segmentation ratio of methanol and water is about50:50, the desorption gradient and volume is successively water—2BV,20%methanol in water—2BV,50%methanol in water—1.8BV, and methanol—2BV. The proper flow rate is1.5BV/h.Thirdly, a suitable HPLC column is chosen from four kinds of columns, which are designed to separate polar compounds, by comparing their degree of separation and resolution. The gradient conditions were then optimized for this column. Results show that NMU C18(4.6×250mm,5μ) is satisfactory for the separation of crude extracts of marine bacteria and actinomycetes. The third gradient condition can separate polar compounds and nonpolar compounds very well. The fingerprints of crude extracts are characterized with HPLC-PDA-ELSD and UPLC-PDA-Q-TOF MS to offer more physico-chemical information.Lastly, the preparative column efficiency is investaged with the dry and wet column packing method, by comparing the reproducibility of chromatographic peak, the repeatability of column packing, the degree of separation and peak symmetry. The feasibility of converting analysis conditions into preparative conditions is preliminarily studied by evalution of column efficiency for both analysis column and preparative column with uracil, nitrobenzene and fluorine. Loading amount and preparation with segmentation are preliminarily studied. Results show that the dry column packing method of is better than the wet method. For the26×460mm column, the proper flow rate is40mL/min and the gradient run time can be calculated by formula4-4with decreased initial concentration of organic solvent. The loading amount of crude extracr should be controlled not to over3grams. |
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