Phosphopeptide enrichment and separation in an affinity microcolumn on a silicon microchip: Comparison of sputtered and wet-deposited TiO2 stationary-phase

We present a microfabricated TiO2 affinity chromatography microcolumn on silicon comprising a deposited TiO2 stationary phase, and demonstrate enrichment and separation of a standard mono-phosphopeptide, and a mixture of non-phosphorylated peptides and multi-phosphopeptides with high capacity. Selective retention of phosphorylated peptides is demonstrated. The TiO2 stationary-phase is formed either with wet deposition or sputtering, and was found to be either amorphous (after sputtering), or crystalline (after sputtering and annealing or after wet deposition). All three methods of deposition (sputtering, sputtering and annealing, liquid deposition) provide enough capacity despite the non-porous nature of the deposited TiO2. The devices can be used several times with reproducible results. The chip design allows an expansion of its capacity (currently 0.4–0.7 μg) by means of increasing the number of parallel microchannels at a constant sample volume. Our approach provides an alternative to off-line extraction tips (with typical capacities of 1–2 μg and sample volumes of 1–10 μL), and to on-chip efforts based on packed bed and frit formats using a standard material for MEMs and standard microfabrication techniques.