Estimation of copper pheophytins, chlorophylls, and pheophytins in mixtures in diethyl ether

Photometric Method for the Quantification of Chlorophylls and Their Derivatives in Complex Mixtures: Fitting with Gauss-Peak Spectra

“…Bréb (strain Greifswald 15) provided by the culture collection of the Botanical Institute, Czech Academy of Sciences, Tře-boň , Č R. Chlorophylls c 1 , c 2 , and their derivatives were prepared from the brown alga Ectocarpus siliculosus (Dillwyn) Lyngbye (strain Port Aransas) provided by D. G. Mü ller, Konstanz University, Federal Republic of Germany. For substituting Mg 2ϩ by Zn 2ϩ or Cu 2ϩ , samples dissolved in 90% methanol were acidified with 10 l concentrated HCl/ml of extract and subsequently saturated with ZnSO 4 or CuSO 4 at about 30°C. These hms-Chl preparations were then transferred to cyclohexane, which was evaporated either in a stream of N 2 or in a vacuum rotary evaporator, and finally the pigments were redissolved in 100% acetone.…”

“…In this paper, we present the Gauss-peak spectra for Quantification of pigments, especially chlorophylls, is a basic prerequisite for the study of many aspects of plant physiology. In most cases, only the main pigments (e.g., Chl a ϩ b in higher plants) 4 are estimated since in unstressed, healthy tissue other chlorophyll derivatives are present only in small amounts. In these cases, quantification can be done by photometry using linear equations like those published by Arnon (1).…”

“…3 E-mail: kuepper@sb-roscoff.fr. 4 Abbreviations used: Chl, chlorophyll; buffer A, buffer used for harvesting cells and preparing thylakoids for green gels, also used for samples, which were subsequently dried and extracted with cyclohexane or acetone; GPS, Gauss peak spectrum, i.e., the series of 4 -5 Gaussian peaks describing a chlorophyll spectrum in the region from 550 to 750 nm; hm, heavy metal; hms, heavy metal substituted; HPLC, high-performance liquid chromatography; Mg substitution, substitution of the natural central ion of Chl, Mg, by heavy metals; Pheo, pheophytin; PUO, parameter used for optimization; SQP, successive quadratic programming; SRCM, spectral reconstitution method (method of Naqvi et al (6)); v ϭ vector of parameters, which are subject to optimization (here: Y0, slope, different chlorophyll derivatives expected in the sample, e.g., Mg-Chl a, Mg-Chl b, Pheo a, and Pheo b in a sample of an unstressed green plant); wldev, wavelength deviation; parameter in the GPS method for the estimation of chlorophylls. …”

“…In most cases, only the main pigments (e.g., Chl a ϩ b in higher plants) 4 are estimated since in unstressed, healthy tissue other chlorophyll derivatives are present only in small amounts. In these cases, quantification can be done by photometry using linear equations like those published by Arnon (1).…”

“…The first such method was published in 1949 by Arnon (1) for the estimation of Chl a ϩ b. This method was later adopted for the estimation of hms-Chls (4,5). There have always been some inherent obstacles when using this traditional method, limiting its accuracy.…”