Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) ; Processo FAPESP: 12/15877-7 ; It is now recognized that analytical chemistry must also be a target for green principles, in particular chromatographic methods which typically use relatively large volumes of hazardous organic solvents. More generally, high performance liquid chromatography (HPLC) is employed routinely for quality control of complex mixtures in various industries. Acetonitrile and methanol are the most commonly used organic solvents in HPLC, but they generate an impact on the environment and can have a negative effect on the health of analysts. Ethanol offers an exciting alternative as a less toxic, biodegradable solvent for HPLC. In this work we demonstrate that replacement of acetonitrile with ethanol as the organic modifier for HPLC can be achieved without significantly compromising analytical performance. This general approach is demonstrated through the specific example analysis of a complex plant extract. A benchmark method employing acetonitrile for the analysis of Bidens pilosa extract was statistically optimized using the Green Chromatographic Fingerprinting Response (GCFR) which includes factors relating to separation performance and environmental parameters. Methods employing ethanol at 30 and 80 C were developed and compared with the reference method regarding their performance of separation (GCFR) as well as by a new metric, Comprehensive Metric to Compare Liquid Chromatography Methods (CM). The fingerprint with ethanol at 80 C was similar to or better than that with MeCN according to GCFR and CM. This demonstrates that temperature may be used to replace harmful solvents with greener ones in HPLC, including for solvents with significantly different physiochemical properties and without loss in separation performance. This work offers a general approach for the chromatographic analysis of complex samples without compromising green analytical chemistry principles. (C) 2014 Published by Elsevier B.V.