Open AccessTime‐resolved SAXS measurements facilitated by online HPLC buffer exchange
Author(s) -
Jensen Malene Hillerup,
Toft Katrine Nørgaard,
David Gabriel,
Havelund Svend,
Pérez Javier,
Vestergaard Bente
Publication year - 2010
Publication title -
journal of synchrotron radiation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.172
H-Index - 99
ISSN - 1600-5775
DOI - 10.1107/s0909049510030372
Subject(s) - small angle x ray scattering , scattering , biological system , chemistry , process (computing) , buffer (optical fiber) , chromatography , materials science , chemical physics , analytical chemistry (journal) , computer science , physics , optics , biology , telecommunications , operating system
Small‐angle X‐ray scattering (SAXS) is a powerful technique to structurally characterize biological macromolecules in solution. Heterogeneous solutions are inherently challenging to study. However, since SAXS data from ideal solutions are additive, with careful computational analysis it may be possible to separate contributions from individual species present in solution. Hence, time‐resolved SAXS (TR‐SAXS) data of processes in development can be analyzed. Many reported TR‐SAXS results are initialized by a sudden change in buffer conditions facilitated by rapid mixing combined with either continuous or stopped flow. In this paper a method for obtaining TR‐SAXS data from systems where the reaction is triggered by removal of a species is presented. This method is based on fast buffer exchange over a short desalting column facilitated by an online HPLC (high‐performance liquid chromatography) connected to the SAXS sample cell. The sample is stopped in the sample cell and the evolving reaction is followed. In this specific system the removal of phenol initiates a self‐association process of long‐acting insulin analogues. For this experiment, data were collected in time series while varying concentrations. The method can be generally applied to other systems where removal of a species or other changes in experimental conditions trigger a process.