Automated phosphopeptide enrichment prior to MS analysis by means of Immobilized Metal Affinity Chromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC) has been probed with packed columns. We compared POROS-Fe 3+ and TiO 2 (respectively IMAC and MOAC media), using a simple mixture of peptides from casein–albumin and a complex mixture of peptides isolated from mouse liver. With theses samples, selectivity of POROS-Fe 3+ and TiO 2 were pH dependant. In the case of liver extract, selectivity increased from 12–18% to 58–60% when loading buffer contained 0.1M acetic acid or 0.1M trifluoroacetic acid, respectively. However, with POROS-Fe 3+ column, the number of identifications decreased from 356 phosphopeptides with 0.1M acetic acid to 119 phosphopeptides with 0.1M TFA. This decrease of binding capacity of POROS-Fe 3+ was associated with strong Fe 3+ leaching. Furthermore, repetitive use of IMAC-Fe 3+ with the 0.5M NH 4 OH solution required for phosphopeptide elution induced Fe 2 O 3 accumulation in the column. By comparison, MOAC columns packed with TiO 2 support do not present any problem of stability in the same conditions and provide a reliable solution for packed column phosphopeptide enrichment.