Bai Yingchen, Wu Fengchang, Xing Baoshan, Meng Wei, Shi Guolan, Ma Yan, Giesy John P
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China.
Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA, 01003, USA.
Sci Rep. 2015 Mar 4;5:8723. doi: 10.1038/srep08723.
XAD-8 adsorption technique coupled with stepwise elution using pyrophosphate buffers with initial pH values of 3, 5, 7, 9, and 13 was developed to isolate Chinese standard fulvic acid (FA) and then separated the FA into five sub-fractions: FApH3, FApH5, FApH7, FApH9 and FApH13, respectively. Mass percentages of FApH3-FApH13 decreased from 42% to 2.5%, and the recovery ratios ranged from 99.0% to 99.5%. Earlier eluting sub-fractions contained greater proportions of carboxylic groups with greater polarity and molecular mass, and later eluting sub-fractions had greater phenolic and aliphatic content. Protein-like components, as well as amorphous and crystalline poly(methylene)-containing components were enriched using neutral and basic buffers. Three main mechanisms likely affect stepwise elution of humic components from XAD-8 resin with pyrophosphate buffers including: 1) the carboxylic-rich sub-fractions are deprotonated at lower pH values and eluted earlier, while phenolic-rich sub-fractions are deprotonated at greater pH values and eluted later. 2) protein or protein-like components can be desorbed and eluted by use of stepwise elution as progressively greater pH values exceed their isoelectric points. 3) size exclusion affects elution of FA sub-fractions. Successful isolation of FA sub-fractions will benefit exploration of the origin, structure, evolution and the investigation of interactions with environmental contaminants.
开发了XAD-8吸附技术，并结合使用初始pH值为3、5、7、9和13的焦磷酸盐缓冲液进行分步洗脱，以分离中国标准富里酸（FA），然后将FA分别分离为五个亚组分：FApH3、FApH5、FApH7、FApH9和FApH13。FApH3至FApH13的质量百分比从42%降至2.5%，回收率在99.0%至99.5%之间。较早洗脱的亚组分含有比例更高的极性和分子量更大的羧基，而较晚洗脱的亚组分含有更多的酚类和脂肪族成分。使用中性和碱性缓冲液可富集类蛋白成分以及含无定形和结晶聚亚甲基的成分。三种主要机制可能影响腐殖质成分用焦磷酸盐缓冲液从XAD-8树脂中分步洗脱，包括：1）富含羧基的亚组分在较低pH值下被去质子化并较早洗脱，而富含酚类的亚组分在较高pH值下被去质子化并较晚洗脱。2）随着逐步升高的pH值超过其等电点，蛋白质或类蛋白成分可通过分步洗脱被解吸和洗脱。3）尺寸排阻影响FA亚组分的洗脱。成功分离FA亚组分将有助于探索其起源、结构、演化以及与环境污染物相互作用的研究。
