Detection Orthogonality in Macromolecular Separations. 2: Exploring Wavelength Orthogonality and Spectroscopic Invisibility Using SEC/DRI/UV/FL.

We continue herein the exploration of detector orthogonality in size-based macromolecular separations. Previously [5], the sensitivity of viscometric detection was juxtaposed to that of differential refractometry (DRI) and light scattering (LS, both static and dynamic), and it was shown that viscometry is a truly orthogonal detection method to both DRI and LS. Here, via the size-exclusion chromatography (SEC) analysis of blends of polystyrene and poly(methyl methacrylate), we demonstrate the orthogonality of DRI to UV detection and, within the UV region of the electromagnetic spectrum, we also explore the phenomenon of "wavelength orthogonality:" Analytes observable by one detection method are shown to be spectroscopically invisible to another method, or even to the same detection method when operating at a different wavelength. While generally focusing on blends of analytes of different molar masses (different sizes in solution), we also investigate the less-explored case of blends of coeluting analytes (same sizes in solution) where detector orthogonality can inform one's knowledge of whether or not coelution has occurred. Finally, by incorporating a fluorescence (FL) detector into the experimental set-up, we demonstrate not only its orthogonality to DRI detection but also its sensitivity to the presence of even minor (≈ 1%) fluorescent components in a sample. We hope the present experiments assist in understanding the complementarity of different spectroscopic detection methods and also help highlight the potential role of FL detection, a method which has been largely overlooked in macromolecular separation science.