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

Polymer Journal volume 55, pages 239–251 (2023)Cite this article

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Abstract

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.

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Acknowledgements

The authors thank AJE (www.aje.com) for English language editing.

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Authors and Affiliations

  1. Department of Organic Materials Science, Graduate School of Organic Materials Science, Yamagata University, 4-3-16, Jonan, Yonezawa, 992-8510, Japan

    Yoshinori Matsumoto, Kayo Ueda, Atsushi Narumi & Seigou Kawaguchi

  2. TOSOH ARC., 1-8, Kasumi, Yokkaichi, 510-8540, Japan

    Yoshinori Matsumoto

  3. Faculty of Engineering, Yamagata University, 4-3-16, Jonan, Yonezawa, 992-8510, Japan

    Moriya Kikuchi

  4. Institute for the Promotion of General Graduate Education, Yamagata University, 4-3-16, Jonan, Yonezawa, 992-8510, Japan

    Kazushi Enomoto

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  1. Yoshinori Matsumoto
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Correspondence to Moriya Kikuchi or Seigou Kawaguchi.

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Matsumoto, Y., Kikuchi, M., Ueda, K. et al. Highly reliable determination of the interdetector delay volume in SEC-MALS for precise characterization of macromolecules having narrow and broad molar mass distributions. Polym J 55, 239–251 (2023). https://doi.org/10.1038/s41428-022-00744-7

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