Control method for integrity of continuous beds

Monoliths are considered to be the latest version of stationary phases for protein chromatography. They are now becoming popular since they exhibit reduced peak broadening due to low mass transfer resistances. A weak point of monoliths may be an inhomogeneous bed, especially when charged groups are present during co-polymerization. This becomes apparent when a protein solution is loaded onto such a monolith. A biphasic breakthrough curve differing from the sigmoidal-shaped one, is observed. Two error functions were used to approximate the breakthrough curve. From these parameters an estimate of the fraction of flow passing through the homogeneously packed part of the bed as well as the inhomogeneously one was derived. This flow pattern was also confirmed by perfusing ferritin through an inhomogeneous monolith and comparing it to an intact one. Electron microscopy and mercury porosimetry did not indicate any abnormalities in the bed.