Open tubular columns are primarily used in gas chromatography, and they were regarded as the best choice to achieve high separation efficiency in gas chromatography. Substances diffuse much slower in liquids than in gases. An open tubular liquid chromatography column must have a small inner diameter to achieve high separation efficiency. We are one of the pioneers that explored narrow open tubular liquid chromatography with 2 μm inner diameter capillaries. Based on previous high efficiency separation of 11 fluorescently derivatized amino acids, we improved the column preparation and greatly enhanced the separation efficiency of the narrow open tubular column. This thesis focuses on optimizing the narrow open tubular column for high efficiency separation, high speed separation, high throughput separation and coupling it with mass spectrometer for single cell bottom-up proteomics.