Quantifying chlorophylls in melanic lichens: the necessity of separating the absorbance of melanin and chlorophyll

This study investigates extraction and quantification techniques for chlorophylls (Chl) in melanic lichens, with an emphasis on distinguishing between Chl and melanin absorbance during spectrophotometric assessments. We compared various extraction protocols, involving solvents such as dimethyl sulfoxide (DMSO) and acetone, and methods including intact thalli extraction, mortar grinding, and ball mill pulverizing. Three correction methods for melanic absorbance were also compared. Our findings indicated that DMSO was superior for Chl extraction compared to acetone, and differences in efficiency among the DMSO methods were minor. Correction for co-extracted melanin was deemed vital for accurate Chl quantification. The use of a C18 minicolumn was found to be effective for separating Chl and melanic pigments, providing reliable measurements of total Chl and Chl a/b-ratios. This method offers a simple cost-effective approach for Chl quantification in extracts containing a mix of Chl and other red light-absorbing pigments.