Separation Of Phenolic Acids From Oat Bran And Sugarcane Skin By PH-Zone-Refining Countercurrent Chromatography

In our country, the resources of oat bran and sugarcane skin are very abundant. In order to further development and comprehensive utilization of its potential value, the effective components from oat bran and sugarcane skin should be deeply studied. As a new separation method, the difference of pH-zone-refining countercurrent chromatography with HSCCC is that the separation is according to the pKa values and hydrophobility of analyte. This method has been widely used to the separation of amino acid derivatives, peptide derivatives, alkaloids, phenolic acids, saponins. In this article, phenolic acids had been separated from oat bran and sugarcane skin by pH-zone-refining countercurrent chromatography.In this thesis, a general separation method of standards phenolic acids using pH-zone-refining counter-current chromatography was established and used to the separation of crude sample of oat bran; the optimized extraction method and pH-zone-refining counter-current chromatography method were established. The main research results were as follows:1. A general separation method of seven standard phenolic acids using pH-zone-refining counter-current chromatography was established. The optimum liquid-liquid system is MTBE-ACN-water (4.75:0.25:5) with TFA (3 mM) in the organic stationary phase and NH4OH (3 mM) in the aqueous mobile phase. As a result, the fractionation of seven standard phenolic acids including syringic acid,4-hydroxyphenylacetic acid, vanillic acid, caffeic acid, P-hydroxybenzoic acid, ferulic acid and P-coumaric acid have been obtained with the purities of 97.0%,67.3%,96.9%,82.4%,95.9%,97.2% and 91.0%, respectively.2. Then 1.22g of pretreated sample using the previous optimized conditions were well separated, and three phenolic acids including 49.5 mg of syringic acid,109.2 mg of P-coumaric acid and 184.5 mg of ferulic acid were obtained with high purities of 95.2%,93.0%, and 91.8%, respectively. The trace caffeic acid and other phenolic acids were also concentrated in the mixing zone.3. The optimized extraction methods of suagarcane skin were established. The extraction method of alkaline hydrolysis was selected after compared with the solvent extraction method, ultrasonic assisted extraction, microwave assisted extraction. The powder of suagarcane skin was extracted by 2 M NaOH at the temperature of 60℃ for 24 h by stirring. The mixture was acidified to pH 2 by adding drops of 6 M HC1. Then the aqueous solution was extracted twice separately with 400 ml of ethyl acetate. The organic fractions were concentrated under reduced pressure in a rotary evaporator.4. The separation method of sugarcane skin crude sample using pH-zone-refining counter-current chromatography was established. The optimized solvent system was MTBE-ACN-water (4:1:5) with TFA (5 mM) in the organic stationary phase and NH4OH (5 mM) in the aqueous mobile phase. As a result, p-coumaric acid was well separated with the purity of 95.99% from 1.00mg of crude sample while other phenolic acids were also concentrated during the process.