Significant calibration of the SAXS instrumentation is applied prior to data collection. Investigators should be aware of four important calibration procedures which will affect all data sets.
The incident beam orientation, sample position, and detector orientation must all be accurately defined in order to calculate scattering plots of Intensity versus q. This is typically done through the collection and analysis of a crystalline powder pattern. Inaccuracy in this calibration will result in blurred SAXS curves where sharp peaks are broadened and the small q scattering may have larger variation.
The incident X-ray wavelength is calibrated typically by measuring absorbance from metal filters with fluorescence near an electron orbital edge. Inaccuracy in wavelength leads to shifted and stretched SAXS profiles with peaks occurring at an alternate apparent q value.
The beamstop and other shadows blocking scattering from the beamline to the detector are masked out. Inaccuracy in defining these regions will lead to large drops in intensity at small q near the beamstop. If the mask is too large, valuable low q data may be obscured.
A solute of known molecular weight and concentration is collected to enable plotting data on an absolute scale. This calibration can be valuable for calculating molecular weight when the concentration of the macromolecule is known. However the scattering contrast between buffer and solute must be considered relative to the calibrant. Including a calibrant on the sample plate is an alternative. These calibration files are readily available if desired.
The incident beam orientation, sample position, and detector orientation must all be accurately defined in order to calculate scattering plots of Intensity versus q. This is typically done through the collection and analysis of a crystalline powder pattern. Inaccuracy in this calibration will result in blurred SAXS curves where sharp peaks are broadened and the small q scattering may have larger variation.
The incident X-ray wavelength is calibrated typically by measuring absorbance from metal filters with fluorescence near an electron orbital edge. Inaccuracy in wavelength leads to shifted and stretched SAXS profiles with peaks occurring at an alternate apparent q value.
The beamstop and other shadows blocking scattering from the beamline to the detector are masked out. Inaccuracy in defining these regions will lead to large drops in intensity at small q near the beamstop. If the mask is too large, valuable low q data may be obscured.
A solute of known molecular weight and concentration is collected to enable plotting data on an absolute scale. This calibration can be valuable for calculating molecular weight when the concentration of the macromolecule is known. However the scattering contrast between buffer and solute must be considered relative to the calibrant. Including a calibrant on the sample plate is an alternative. These calibration files are readily available if desired.
