Molar mass profiling of synthetic polymers by free-solution capillary electrophoresis of DNA-polymer conjugates

The molar mass distribution of a polymer sample is a critical determinant o f its material properties and is generally analyzed by gel permeation chrom atography or more recently, by MALDI-TOF mass spectrometry, We describe her e a novel method for the determination of the degree of polymerization of p olydisperse, 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 cov alent conjugation of a polydisperse, uncharged polymer sample (PEG) to a mo nodisperse, 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 hydro dynamic drag. The balance of electromotive and hydrodynamic forces enables rapid, high-resolution separation of the DNA-polymer conjugates as a functi on of the size of the uncharged PEG tail. This provides a profile of the mo lar mass distribution of the original polymer sample that can be detected b y laser-induced fluorescence through excitation of the dye-labeled DNA. We call this method free solution conjugate electrophoresis (FSCE), Theory-bas ed analysis of the resulting electrophoresis data allows precise calculatio n of the degree of polymerization of the PEG portion of each conjugate mole cule. Knowledge of the molecular mass of the uncharged polymer's repeat uni t allows for direct calculation of the molar mass averages as well as sampl e polydispersity index. The results of these analyses are strikingly remini scent of MALDI-TOF spectra taken of the same PEG samples. PEG samples of 3. 4-, 5-, and 20kDa nominal average molar mass were analyzed by FSCE and MALD I-TOF; the values of the molar mass averages, M-w and M-n, typically agree to within 5%, Measurements and molar mass calculations are performed withou t any internal standards or calibration. Moreover, when DNA-polymer conjuga te analysis is performed in a chip-based electrophoresis system, separation is complete in less than 13 min, FSCE offers an alternative to MALDI-TOF f or the characterization of uncharged, water-soluble polymers that can be un iquely conjugated to DNA.