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.