Experimental methods in chemical engineering: High performance liquid chromatography—HPLC

High performance liquid chromatography (HPLC) identifies compounds in a sample and quantifies their concentration. The instrument consists of an injector that introduces a µL sample to a mobile phase that carries the analytes across a column filled with an adsorbing substance. The analytes adsorb and desorb at different rates as they flow down the column and are thereby separated. Detectors record a signal proportional to the concentration of the analyte in the mobile phase. Laboratories apply modern HPLC systems for research and development, quality control, safety, and validation. Academia and industry resort to HPLC to purify, identify, and characterize a wide variety of molecules at all stages of a process. Developing an HPLC analytical method is a long and laborious process requiring standards, calibration curves, derivatization, and multiple tests to identify an appropriate column, stationary phase, flow rate, temperature, and mobile phase. Optimal operating conditions maintain a good resolution (high signal-to-noise ratio and low peak overlap) while minimizing analysis time. Web of Science Core Collection indexed 8900 articles in 2019, which is relatively few compared to X-ray and scanning electron microscopy. However, it is such an ubiquitous technique and we estimate that the number of articles that report it in the experimental section is an order of magnitude higher. The bibliometric analysis of the keywords identified 4 research clusters: phenolics and flavonoids, extraction and pharmacokinetics, in vitro and oxidative stress, and optimization. Here, we detail a walkthrough for the basic steps required to develop a chromatographic method.