New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton

The Spectral Determination of Chlorophylls a and b, as well as Total Carotenoids, Using Various Solvents with Spectrophotometers of Different Resolution*

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.

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

A set of equations for determining chlorophyll a (Chl a) and accessory chlorophylls b, c
 2
 , c
 1
  + c
 2 and the special case of Acaryochloris marina, which uses Chl d as its primary photosynthetic pigment and also has Chl a, have been developed for 90% acetone, methanol and ethanol solvents. These equations for different solvents give chlorophyll assays that are consistent with each other. No algorithms for Chl c compounds (c
 2
 , c
 1
  + c
 2) in the presence of Chl a have previously been published for methanol or ethanol. The limits of detection (and inherent error, ± 95% confidence limit), for chlorophylls in all organisms tested, was generally less than 0.1 µg/ml. The Chl a and b algorithms for green algae and land plants have very small inherent errors (< 0.01 µg/ml). Chl a and d algorithms for Acaryochloris
 marina are consistent with each other, giving estimates of Chl d/a ratios which are consistent with previously published estimates using HPLC and a rarely used algorithm originally published for diethyl ether in 1955. The statistical error structure of chlorophyll algorithms is discussed. The relative error of measurements of chlorophylls increases hyperbolically in diluted chlorophyll extracts because the inherent errors of the chlorophyll algorithms are constants independent of the magnitude of absorbance readings. For safety reasons, efficient extraction of chlorophylls and the convenience of being able to use polystyrene cuvettes, the algorithms for ethanol are recommended for routine assays of chlorophylls. The methanol algorithms would be convenient for assays associated with HPLC work.

https://www.researchgate.net/profile/Raymond_J_Ritchie/publication/7019194_Consistent_Sets_of_Spectrophotometric_Chlorophyll_Equations_for_Acetone_Methanol_and_Ethanol_Solvents/links/0d1c84f9233a3012fd000000.pdf

Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents.

Eleven species of marine planktonic representatives of the Chlorophyceae, Chrysophyceae, Bacillariophyceae, Dinophyceae and Myxophyceae have been analyzed for their chemical composition. All specie...

On the Chemical Composition of Eleven Species of Marine Phytoplankters

Chlorophylls are the green pigments in plants some of which, and perhaps all, participate in photosynthesis. An understanding of their nature, genesis, transformation, and function in plants requires that they and their near chemical relatives be recognized qualitatively and oftentimes be determined quantitatively. In addition to the use of analytical procedures for the identification and determination of the chlorophylls as participants in plant physiological processes, these methods may be used in various other fields of science and technology some of which are genetics, plant nutrition, certain aspects of pure chemistry, crop production of land and sea, medicine, pharmaceuticals, food technology, and paleontology.

Chlorophylls: Analysis in Plant Materials

riboflavin, thiamine, and carotene content of leaf blades was not statistically significant over a period of six weeks. In midribs and roots, the concentration of each of the vitamins decreased as the plants grew larger and the decrease was statistically significant for each vitamin except carotene, in midribs, and thiamine, in roots. Distribution of the whole plant content of carotene, riboflavin, and thiamine among plant organs did not change appreciably as plant dry weight increased, but there was a marked change in the distribution of ascorbic acid which rather closely followed the distribution of plant dry matter. In all experiments the total amount of each vitamin per plant was positively and significantly correlated with the total amount of plant dry matter. Increase in the total amount of vitamin per plant per unit increase in plant dry matter was characteristic for each vitamin and was not influenced by the environmental conditions under which the plants were grown. Regression equations derived for the estimation of the amount of each of the vitamins per plant from plant dry weight were applicable to plants weighing from 2 grams to 69 grams. LITERATURE CITED

Allen Benitez

Papers

Chlorophylls: Analysis in Plant Materials

The Effect of Temperature on the Conversion of Protochlorophyll to Chlorophyll a in Etiolated Barley Leaves.