The importance of establishing robust and reliable analytical methods is of paramount importance in today’s life science research and the (bio)pharmaceutical industry. To meet the required quality specifications imposed by authorities, analytical methods that have proven to yield consistent results are essential. Time and resource consuming processes where these methods need to be re-evaluated and validated, should be avoided at all cost [1]. Liquid chromatography, either coupled with UV detection or mass spectrometry has a prominent position within biomarker discovery and quality control workflows. Among other factors, the quality of the LC column has a significant impact on the data reproducibility and method robustness. LC columns are typically fabricated by packing spherical silica particles into a cylindrical column. Even though column technology has improved enormously in the past decades, batch-to-batch repeatability is still a critical issue that can have a serious impact on LC workflow robustness. Aside batch-to-batch variations (particle size distribution and chemical composition) of the silica material, the packing process itself introduces a certain degree of heterogeneity, preventing the fabrication of multiple LC columns with an identical stationary phase backbone morphology.
By using an entirely different LC column fabrication process, where nanometer precision 2D designs are transferred onto silicon wafers and transformed into a uniform array of superficially porous silicon pillars, PharmaFluidics brings an extremely robust alternative to the LC column market, the micro Pillar Array Column format (μPAC™). Apart from eliminating virtually any column to column variability, precise positioning of these 5 μm diameter silicon pillars creates a stationary phase support that introduces minimal dispersion (or dilution of the samples) into the separation process [2, 3]. These columns can also be operated at LC pump pressures that are significantly lower than what is needed to operate the current state-of-the-art in packed bed capillary flow LC columns (sub 2 μm particles), hereby reducing the shear force on LC pump components and positively affecting their lifetime.
The main goal of this study is to compare the column-to-column reproducibility of the μPAC™ capLC column format to state-of-the art commercially available packed bed column alternatives. The basic performance characteristics of the μPAC™ capLC have already been discussed in previous technical and application notes [4-6]. Reversed phase capillary LC analysis of a protein tryptic digest (Cytochrome C digest) is performed on a series of columns (three column types, n=3), and column-to-column reproducibility is compared in terms of retention, efficiency and peak shape.
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