Size-exclusion chromatography–small-angle neutron scattering system optimized for an instrument with medium neutron flux

Abstract

Biomacromolecular solutions inevitably contain impurities in addition to the target biomacromolecules. This has been a major obstacle to achieving highprecision solution scattering measurements. To overcome this problem, smallangle X-ray scattering (SAXS) coupled with size-exclusion chromatography (SEC-SAXS) has been developed. This method involves injecting the solution eluted by SEC directly into a measurement cell and conducting SAXS measurements during the elution of the target biomacromolecule. This technique has resulted in a paradigm shift in biomacromolecule solution scattering. Currently, the application of the SEC–SAXS system to small-angle neutron scattering (SANS) is being advanced. However, since the target biomacromolecules in the sample solution are not only purified but also diluted by SEC and pass through the sample cell in a short time, this method is being implemented in SANS instruments at high neutron flux. Here, we developed a new type of SEC–SANS system that can operate effectively with a SANS instrument at medium neutron flux. Its key innovation is the design and optimization of a dedicated flow path that allows for the storage of only the target biomolecules eluted from SEC in the sample cell. This innovation enables long-duration measurements, termed the ‘stopping mode’, for SEC samples. Consequently, this method allows for acquiring high-precision solution scattering data for target biomacromolecules, enabling SEC–SANS measurements even with SANS instruments at medium neutron flux.