This paper reports a miniaturized Reverse-Phase High Performance Liquid Chromatography (HPLC) column for chemically hazardous molecules. The mini-column is designed to generate a phase delay for mixed sample molecules by the reverse-phase separation mechanism. The reverse-phase separation is performed by silica-based nano-pore beads (C-18) as stationary phase and a mixture of methanol and DI water as mobile phase. The C-18 beads are packed inside the compact chamber enclosed by a custom built PDMS (Polydimethylsiloxane) housing, equipped with microfluidic channels, inlet/outlet ports, and connectors. The mobile phase and target analytes are controlled by a syringe pump capable of manipulating two different flow rates using LabVIEW interface. The mini-column successfully separates two sample chemical molecules: Acetaminophen and Aspirin. The intensity ratio of two molecules decreases 35% from two different samples collecting at 3 and 8 min.