Zhou Zhengzhen, Guo Laodong
School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 East Greenfield Avenue, Milwaukee, WI 53204, USA.
School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 East Greenfield Avenue, Milwaukee, WI 53204, USA.
J Chromatogr A. 2015 Jun 19;1399:53-64. doi: 10.1016/j.chroma.2015.04.035. Epub 2015 Apr 27.
Colloidal retention characteristics, recovery and size distribution of model macromolecules and natural dissolved organic matter (DOM) were systematically examined using an asymmetrical flow field-flow fractionation (AFlFFF) system under various membrane size cutoffs and carrier solutions. Polystyrene sulfonate (PSS) standards with known molecular weights (MW) were used to determine their permeation and recovery rates by membranes with different nominal MW cutoffs (NMWCO) within the AFlFFF system. Based on a ≥90% recovery rate for PSS standards by the AFlFFF system, the actual NMWCOs were determined to be 1.9 kDa for the 0.3 kDa membrane, 2.7 kDa for the 1 kDa membrane, and 33 kDa for the 10 kDa membrane, respectively. After membrane calibration, natural DOM samples were analyzed with the AFlFFF system to determine their colloidal size distribution and the influence from membrane NMWCOs and carrier solutions. Size partitioning of DOM samples showed a predominant colloidal size fraction in the <5 nm or <10 kDa size range, consistent with the size characteristics of humic substances as the main terrestrial DOM component. Recovery of DOM by the AFlFFF system, as determined by UV-absorbance at 254 nm, decreased significantly with increasing membrane NMWCO, from 45% by the 0.3 kDa membrane to 2-3% by the 10 kDa membrane. Since natural DOM is mostly composed of lower MW substances (<10 kDa) and the actual membrane cutoffs are normally larger than their manufacturer ratings, a 0.3 kDa membrane (with an actual NMWCO of 1.9 kDa) is highly recommended for colloidal size characterization of natural DOM. Among the three carrier solutions, borate buffer seemed to provide the highest recovery and optimal separation of DOM. Rigorous calibration with macromolecular standards and optimization of system conditions are a prerequisite for quantifying colloidal size distribution using the flow field-flow fractionation technique. In addition, the coupling of AFlFFF with fluorescence EEMs could provide new insights into DOM heterogeneity in different colloidal size fractions.
在不同的膜截留分子量和载体溶液条件下，使用不对称流场流分馏（AFlFFF）系统，系统地研究了模型大分子和天然溶解有机物（DOM）的胶体保留特性、回收率和尺寸分布。使用具有已知分子量（MW）的聚苯乙烯磺酸盐（PSS）标准品，来确定它们在AFlFFF系统中通过不同标称截留分子量（NMWCO）的膜时的渗透和回收率。基于AFlFFF系统对PSS标准品的回收率≥90%，确定0.3 kDa膜的实际NMWCO为1.9 kDa，1 kDa膜的实际NMWCO为2.7 kDa，10 kDa膜的实际NMWCO为33 kDa。在膜校准后，使用AFlFFF系统分析天然DOM样品，以确定其胶体尺寸分布以及膜NMWCO和载体溶液的影响。DOM样品的尺寸划分显示，在<5 nm或<10 kDa尺寸范围内存在主要的胶体尺寸级分，这与作为主要陆地DOM组分的腐殖质的尺寸特征一致。通过在254 nm处的紫外吸光度测定，AFlFFF系统对DOM的回收率随着膜NMWCO的增加而显著降低，从0.3 kDa膜的45%降至10 kDa膜的2 - 3%。由于天然DOM大多由较低分子量的物质（<10 kDa）组成，且实际的膜截留值通常大于其制造商标称值，因此强烈推荐使用0.3 kDa膜（实际NMWCO为1.9 kDa）来表征天然DOM的胶体尺寸。在三种载体溶液中，硼酸盐缓冲液似乎能提供最高的回收率和最佳的DOM分离效果。使用大分子标准品进行严格校准和优化系统条件是使用流场流分馏技术定量胶体尺寸分布的前提条件。此外，AFlFFF与荧光EEMs的联用可以为不同胶体尺寸级分中DOM的异质性提供新的见解。
