Evaluation between ultrahigh pressure liquid chromatography and high-performance liquid chromatography analytical methods for characterizing natural dyestuffs

• UHPLC gave more improved resolution than HPLC for the analysis of natural dyes.
• The most suitable UHPLC results were obtained with a BEH Shield column.
• A spectral database was built with the characterization of 85 dye compounds.
• UHPLC/spectral database is an important tool for further research on natural dyes.
• UHPLC provides accurate results to characterize natural dyes in cultural heritage.

An evaluation was undertaken of ultrahigh pressure liquid chromatography (UHPLC) in comparison to high-performance liquid chromatography (HPLC) for characterizing natural dyes in cultural heritage objects. A new UHPLC method was optimized by testing several analytical parameters adapted from prior UHPLC studies developed in diverse fields of research. Different gradient elution programs were tested on seven UHPLC columns with different dimensions and stationary phase compositions by applying several mobile phases, flow rates, temperatures, and runtimes. The UHPLC method successfully provided more improved data than that achieved by the HPLC method. Indeed, even though carminic acid has shown circa 146% higher resolution with HPLC, UHPLC resulted in an increase of 41–61% resolution and a decrease of 91–422% limit of detection, depending on the dye compound. The optimized method was subsequently assigned to analyse 59 natural reference materials, in which 85 different components were ascribed with different physicochemical properties, in order to create a spectral database for future characterization of dyes in cultural heritage objects. The majority of these reference samples could be successfully distinguished with one single method through the examination of these compounds’ retention times and their spectra acquired with a photodiode array detector. These results demonstrate that UHPLC analyses are extremely valuable for the acquisition of more precise chromatographic information concerning natural dyes with complex mixtures of different and/or closely related physicochemical properties, essential for distinguishing similar species of plants and animals used to colour cultural heritage objects.