The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b

Over the last half century, the most frequently used assay for chlorophylls in higher plants and green algae, the Arnon assay [Arnon DI (1949) Plant Physiol 24: 1-15], employed simultaneous equations for determining the concentrations of chlorophylls a and b in aqueous 80% acetone extracts of chlorophyllous plant and algal materials. These equations, however, were developed using extinction coefficients for chlorophylls a and b derived from early inaccurate spectrophotometric data. Thus, Arnon's equations give inaccurate chlorophyll a and b determinations and, therefore, inaccurate chlorophyll a/b ratios, which are always low. This paper describes how the ratios are increasingly and alarmingly low as the proportion of chlorophyll a increases. Accurate extinction coefficients for chlorophylls a and b, and the more reliable simultaneous equations derived from them, have been published subsequently by many research groups; these new post-Arnon equations, however, have been ignored by many researchers. This Minireview records the history of the development of accurate simultaneous equations and some difficulties and anomalies arising from the retention of Arnon's seriously flawed equations.