Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy

The extinction coefficients for chlorophylls a and b in diethylether (Smith, J.H.C. and Benitez, A. (1955) in Modern Methods of Plant Analysis (Paech, K. and Tracey, M.V., eds.), Vol. 4, pp. 143–196, Springer-Verlag, Berlin), used in this paper as primary standards, were verified, to within an error of less than 1%, by magnesium determination using atomic absorbance spectrophotometry. We also report the determination of accurate extinction coefficients for chlorophylls a and b in N,N′-dimethylformamide, methanol or buffered 80% aqueous acetone. Highly purified chlorophylls were used and methods were employed which not only minimize errors due to evaporation of the volatile solvents employed in their estimation but also eliminate variable micro-contamination by chlorophyll degradation products, a potential source of inconsistency between the extinction coefficients obtained in each of these three solvents. Using these new coefficients, expressed as both millimolar and specific coefficients, we have derived new simultaneous equations to obtain chlorophyll concentrations as nmol/ml and μg/ml, respectively. These equations were applied to data obtained with leaf discs from spinach and Flindersia brayleyana extracted with the three specified solvents and to a concentrated solution (in N,N′ -dimethylformamide) of a chlorophyll a + b mixture added to the threesolvent systems. The validity of these equations is proven by the consistency of the chlorophyll determinations and of the chlorophyll a/b ratios. New simultaneous equations, compatible with the equations derived for the threesolvents, are presented for the assay of chlorophylls a and b converted to their cyclic hydroxylactone derivatives by extraction with alkaline pyridine reagent (2.1 M pyridine in 0.35 M NaOH). Most chlorophyll analyses in higher plants, including the chlorophyll content and chlorophyll a/b ratios of plant thylakoids and chlorophyll-protein complexes, have been obtained in 80% aqueous acetone with the much used simultaneous equations of Arnon (Arnon, D.I. (1949) Plant Physiol. 24, 1–15). For this reason we include conversion factors whichcorrect these earlier data and make it compatible with data calculated with the simultaneous equations presented in this paper. The importance of these corrections to the formulation of meaningful models of the photosynthetic apparatus is demonstrated. Our results also indicate that grinding leaf discs with N,N′-dimethylformamide is a more reliable method for extracting all chlorophylls than shaking with this solvent for 24 h.