μPAC™ column performance with peptide standards: Thermo Scientific™ Pierce™ retention time calibration mixture

μPAC™ column performance with peptide standards: Thermo Scientific™ Pierce™ retention time calibration mixture

Introduction

Peptide standards are critical in mass spectrometry based proteomics to ensure optimal and consistent system performance before, during, and after sample analysis. They can be used to assess peptide elution, troubleshoot chromatography, predict retention time, and to demonstrate that the LC-MS platforms are working properly.

As an alternative to classical packed-bed LC columns, PharmaFluidics offers micromachined nano LC chip columns or micro pillar array columns (μPAC™). The inherent high permeability and low ‘on-column’ dispersion obtained by the perfect order of the separation bed makes μPAC™ based chromatography unique in its kind. The peak dispersion originating from heterogeneous flow paths in the separation bed is eliminated (no A-term contributions) and therefore components remain much more concentrated during separation [1]. The freestanding nature of the pillars also leads to much lower backpressure allowing the use of very long columns with exceptional peak capacities [2].

Because of these unique features, column installation, initial operation and performance evaluation have to be adapted to match the column’s properties. In this technical note we describe the use of Thermo Scientic™ Pierce™ Retention Time calibration mixture as an internal retention time standard for bottom-up proteomics experiments performed on a 200 cm long PharmaFluidics μPAC™ column.