Highly reliable determination of the interdetector delay volume in SEC-MALS for precise characterization of macromolecules having narrow and broad molar mass distributions

• Published in: Polymer journal Vol. 55; no. 3; pp. 239 - 251
• Main Authors: Matsumoto, Yoshinori, Kikuchi, Moriya, Ueda, Kayo, Enomoto, Kazushi, Narumi, Atsushi, Kawaguchi, Seigou
• Format: Journal Article
• Language: English
• Published: Tokyo Nature Publishing Group 01.03.2023
• Subjects: Delay; Detectors; Distribution functions; Ethylbenzene; Flow velocity; Gels; Gyration; Light scattering; Macromolecules; Parameters; Polystyrene resins; Reliability; Sensors; Serum albumin; Size exclusion chromatography; Weight
• ISSN: 0032-3896; 1349-0540
• DOI: 10.1038/s41428-022-00744-7

Size exclusion chromatography (SEC) with a multiangle light scattering (MALS) detector is widely used as standard molecular characterization equipment. The interdetector delay volume (IDV) between the MALS and differential refractometer (RI) detectors is an essential instrumental parameter that affects reliability and accuracy. However, a reliable and accurate determination method has not been established to date. This paper presents a method for determining the IDV value. SEC-MALS elution behavior has been thoroughly studied for polystyrene (PSt) standards with weight average molar mass (Mw) ranging from 1.02 × 104 to 109 × 104 g mol−1, ethylbenzene, and bovine serum albumin at different flow rates (U) ranging from 0.1 to 1.0 cm3 min−1 and at different particle sizes (dp) of the porous packing gels ranging from 5 to 30 μm. The apparent IDV values, IDVapp, estimated by superimposing the RI signal on the MALS signal, varied considerably with U, dp, and Mw because of band broadening and turbulence. The heights equivalent to a theoretical plate, H, were estimated for the MALS chromatogram (HMALS) and RI chromatogram (HRI), and they were rationalized as a function of U, Mw, and dp. The IDVapp functions HMALS and ΔH = HMALS − HRI corresponded to the band broadening occurring in SEC columns and the disturbance effects in the connecting tube between the MALS and RI detectors and in both cells, respectively. A reliable IDV was obtained when both the HMALS and ΔH values were minimized, i.e., U → 0 and Mw → 0. The IDV value determined at U = 0 and Mw = 0 was 24% lower than resulting from the currently recommended method. A SEC-MALS setup with the correct IDV value enables one to estimate more accurately not only the Mw and z-average root-mean-square radius of gyration but also the number average molar mass (Mn) and differential weight distribution function for PSts having narrow and broad molar mass distributions.The interdetector delay volumes (IDV) for multiangle light scattering (MALS) and differential refractometer detectors are essential instrumental parameters affecting reliability and accuracy. However, a reliable and accurate determination method has not yet been established. The paper proposes a method for determining the IDV value. An SEC-MALS setup with the correct IDV value provides more accurate estimates of not only the weight average molar mass and z-average mean-square radius of gyration but also the number average molar mass and differential weight distribution function for polymer samples having narrow and broad molar mass distributions.