Asymmetrical flow field-flow fractionation is a versatile chromatographic fractionation method. In combination with at least one detection technique it is used for size-based separation of colloids, biomolecules and polymers. Although often used as pure separation method, a well-elaborated theory is available that allows precise quantification of the translational diffusion coefficient D. Still, current literature suggests different ways to transform this theory into applicable experimental procedures and no “gold standard” for correct data processing exists. While some sources report a direct way to extract diffusion information from the fractogram, others suggest the necessity of an external calibration measurement to obtain the channel width w. In this work, we compare the different approaches and calibration algorithms based on original and literature data using our own open-source AF4 evaluation software. Based on the results, we conclude that available AF4 setups do not fulfill the requirements for absolute measurements of D. We show that the best way to conduct is to consider the area of the channel and D of the calibrant while neglecting the small peak which occurs in the void peak region.

非对称流场-流分离是一种通用的色谱分离方法。与至少一种检测技术结合，可用于基于尺寸的胶体，生物分子和聚合物分离。尽管通常用作纯分离方法，良好的详尽理论是可用，允许平移扩散系数的精确定量d。但是，当前的文献提出了将这种理论转化为适用的实验程序的不同方法，并且不存在用于正确数据处理的“黄金标准”。虽然某些来源报告了直接从分形图中提取扩散信息的方法，但另一些来源建议必须进行外部校准测量以获得通道宽度w。在这项工作中，我们使用我们自己的开源AF4评估软件比较了基于原始数据和文献数据的不同方法和校准算法。根据结果​​，我们得出结论，可用的AF4设置不能满足D绝对测量的要求。我们表明，最佳的传导方式是考虑通道的面积和校准物的D，而忽略在空峰区域中出现的小峰。