TiO2-ZrO2 affinity chromatography polymeric microchip for phosphopeptide enrichment and separation

We fabricated a TiO2-ZrO2 affinity chromatography micro-column on 2 mm PMMA plates, and demonstrated the enrichment and separation of (a) a standard mono-and tetra-phosphopeptide, and (b) phosphopeptides contained in a tryptic digest of beta-Casein. The chromatography column consisted of 32 parallel microchannels with common input and output ports and was fabricated by lithography directly on the polymeric substrate followed by plasma etching (i.e. standard MEMS processing) and sealed with lamination. The liquid deposited TiO2-ZrO2 stationary phase was characterized by X-ray diffraction and was found to be mostly TiO2 and ZrO2 in crystalline phases. Off-chip UV detection and MALDI MS identification of the separated effluents were used. The chip had a capacity of > 1.4 mu g (0.7 nmol) of a prototype mono-phosphopeptide and a recovery of 94 +/- 3%, and can be used with small samples (less than 0.1 mu L depending on the syringe pump used). The chip design allows an expansion of its capacity by means of increasing the number of parallel microchannels at a constant sample volume. Our approach provided an alternative to off-line extraction tips (with typical capacities of 1-2 mu g and sample volumes of 1-10 mu L), and to on-chip efforts based on packed bed and frit formats.