Vreeland W N, Desruisseaux C, Karger A E, Drouin G, Slater G W, Barron A E
Department of Chemical Engineering, Northwestern University, Evanston, Illinois 60208, USA.
Anal Chem. 2001 Apr 15;73(8):1795-803. doi: 10.1021/ac001380+.
The molar mass distribution of a polymer sample is a critical determinant of its material properties and is generally analyzed by gel permeation chromatography or more recently, by MALDI-TOF mass spectrometry. We describe here a novel method for the determination of the degree of polymerization of polydisperse, uncharged, water-soluble polymers (e.g., poly(ethylene glycol) (PEG)), based upon single-monomer resolution of DNA-polymer conjugates by free-solution capillary electrophoresis. This is accomplished by end-on covalent conjugation of a polydisperse, uncharged polymer sample (PEG) to a monodisperse, fluorescently labeled DNA oligomer, followed by electrophoretic analysis. The monodisperse, charged DNA "engine" confers to each conjugate an equal amount of electromotive force, while the varying contour lengths of the uncharged, polydisperse polymers engender different amounts of hydrodynamic drag. The balance of electromotive and hydrodynamic forces enables rapid, high-resolution separation of the DNA-polymer conjugates as a function of the size of the uncharged PEG tail. This provides a profile of the molar mass distribution of the original polymer sample that can be detected by laser-induced fluorescence through excitation of the dye-labeled DNA. We call this method free solution conjugate electrophoresis (FSCE). Theory-based analysis of the resulting electrophoresis data allows precise calculation of the degree of polymerization of the PEG portion of each conjugate molecule. Knowledge of the molecular mass of the uncharged polymer's repeat unit allows for direct calculation of the molar mass averages as well as sample polydispersity index. The results of these analyses are strikingly reminiscent of MALDI-TOF spectra taken of the same PEG samples. PEG samples of 3.4-, 5-, and 20-kDa nominal average molar mass were analyzed by FSCE and MALDI-TOF; the values of the molar mass averages, Mw and Mn, typically agree to within 5%. Measurements and molar mass calculations are performed without any internal standards or calibration. Moreover, when DNA-polymer conjugate analysis is performed in a chip-based electrophoresis system, separation is complete in less than 13 min. FSCE offers an alternative to MALDI-TOF for the characterization of uncharged, water-soluble polymers that can be uniquely conjugated to DNA.
聚合物样品的摩尔质量分布是其材料性能的关键决定因素，通常通过凝胶渗透色谱法进行分析，或者最近通过基质辅助激光解吸电离飞行时间质谱法（MALDI - TOF）进行分析。我们在此描述一种新方法，用于测定多分散、不带电荷的水溶性聚合物（例如聚乙二醇（PEG））的聚合度，该方法基于通过自由溶液毛细管电泳对DNA - 聚合物共轭物进行单单体分辨率分析。这是通过将多分散、不带电荷的聚合物样品（PEG）与单分散、荧光标记的DNA寡聚物进行端对端共价偶联，然后进行电泳分析来实现的。单分散、带电荷的DNA“引擎”赋予每个共轭物等量的电动势，而不带电荷的多分散聚合物的不同轮廓长度产生不同量的流体动力学阻力。电动势和流体动力学力的平衡使得能够根据不带电荷的PEG尾部大小快速且高分辨率地分离DNA - 聚合物共轭物。这提供了原始聚合物样品的摩尔质量分布图谱，可通过激发染料标记的DNA的激光诱导荧光进行检测。我们将此方法称为自由溶液共轭电泳（FSCE）。基于理论对所得电泳数据进行分析，可以精确计算每个共轭分子中PEG部分的聚合度。了解不带电荷聚合物重复单元的分子量可以直接计算摩尔质量平均值以及样品多分散指数。这些分析结果与对相同PEG样品进行MALDI - TOF光谱分析的结果惊人地相似。通过FSCE和MALDI - TOF对标称平均摩尔质量为3.4 kDa、5 kDa和20 kDa的PEG样品进行了分析；摩尔质量平均值Mw和Mn的值通常在5%以内一致。测量和摩尔质量计算无需任何内标或校准即可进行。此外，当在基于芯片的电泳系统中进行DNA - 聚合物共轭物分析时，分离在不到13分钟内即可完成。对于可与DNA独特共轭的不带电荷的水溶性聚合物的表征，FSCE为MALDI - TOF提供了一种替代方法。
