We used chromatography modeling software to assist in HPLC method development, with the goal of enhancing separations through the exclusive use of gradient time and column temperature. We surveyed nine stationary phases for their utility in pigment purification and natural sample analysis. For purification, a complex algal matrix was separated on an efficient monomeric column, from which partially purified fractions were collected and purified on polymeric columns that exaggerated resolution between pigments of interest. Additionally, we feature an HPLC method that is simple, fast, demonstrates excellent transferability and is ideal for quantitative analysis of pigments in dilute natural water samples.