BACKGROUND: The fouling impact of selected fouling species was assessed by utilising confocal scanning laser microscopy (CSLM) to image a packed chromatographic bed during operation. A custom-made flow cell was packed with Q Sepharose FF and loaded with partially clarified E. coli homogenate. Selective, multicoloured fluorescent dyes were used to label a bovine serum albumin (BSA) test protein (Cy5.5), dsDNA (PicoGreen) and host cell proteins (HCPs) (Cy3). The fouling caused by the various fluorescently labelled components was visualised as a result of the fluorescence emitted by the PicoGreen-labelled dsDNA and the Cy3-labelled protein in the foulant stream, and by testing the adsorptive capacity of a test protein (BSA) onto the resin prior to and post-fouling as well as following the application of a common CIP procedure. RESULTS: Values for the effective diffusivity of BSA (De) were derived from the confocal images and the fouling impact was assessed by comparing D. values obtained from different fouling scenarios. Under the most extreme conditions examined, fouling caused a 20% reduction in capacity compared to a fresh bed. BSA diffusivity did not appear to be affected by the fouling conditions studied. Sequential CIP using 15 CVs of 1 mol L-1 NaCl then 15 CVs of 1 mol L-1 NaOH was shown to be effective in removing nucleic acids and HCPs. Subsequent BSA adsorption showed that the CIP regime successfully restored the column capacity to its original value. In contrast, 15 CVs of 1 mol L-1 NaCl were ineffective in removing dsDNA but substantially removed HCPs. CONCLUSION: CSLM was demonstrated to be a useful tool for visualising fouling mechanisms. Comparing the results obtained by this technique using different modes of chromatographic operation provided insights into the fouling characteristics of finite baths versus packed beds. (c) 2007 Society of Chemical Industry.