Chlorophylls and derivatives

Several analytical methods are available to quantify chlorophylls and choice depends on the information needed. For quality control in industries and legislation attendance, simple and cost-effective methods represent widely used problem-solving approaches. For research purposes, more sensitive and precise methods are necessary to identify chlorophylls and derivatives simultaneously and individually. 

Due to the high mass, low volatility, and thermal instability of chlorophylls and derivatives, molecular weight determination by electron impact (El) MS is not recommended. Desorption-ionization MS techniques such as chemical ionization, secondary ion MS, fast-atom bombardment (FAB), field, plasma- and matrix-assisted laser desorption have been very effective for molecular ion detection in the characterization of tetrapyrroles. These techniques do not require sample vaporization prior to ionization and they are effective tools for allomerization studies. 

All the analytical methods mentioned to separate, identify, and quantify chlorophylls and derivatives consume time, money, and samples. As alternatives, industries have been employing non-destructive methods for surface color measurements that are not only indirectly related to chlorophyll content, but may also estimate the pigments directly in tissues, leaving the sample intact and enabling serial analyses in a relatively short time. Eood color affects consumer acceptance and is an important criterion for quality control. Color vision is a complex phenomenon that depends on both the total content and number of pigments and also on absorption, reflectance and emission spectra of each compound present. 

W. H. Eddy, Chlorophyll and Derivatives. Strong Cobb, Cleveland, Ohio, 1953. 

Table 14.4. Infrared Absorption Bands of Chlorophylls and Derivatives Frequencies Corrected (cm ) (after Holt and Jacobs, 1955). (Marks, 1969.)... 

Oil suitable for deodorization must be free of impurities that can undergo rapid heat degradation and which can impair the flavor and color stability. The following are notable phosphatides, which should not exceed 5 ppm as phosphorus equivalent chlorophyll and derivatives, which should be essentially undetectable, and nickel, which should be below 0.5 ppm. Further, the prooxidant metals iron and copper should not exceed 0.1 and 0.01 ppm, respectively (Evans et a/., 1951). 

Chlorophylls and phaeophytins are fat-soluble pigments. Chloro-phyhdes and phaeophorbides are hydrophUic pigments soluble in water due to the absence of phytol. The colours of chlorophylls and derived products are given in Table 9.8. 

Nuclear magnetic resonance (NMR) spectroscopy, MS, and infrared (IR) spectroscopy have been used successfully in the structural elucidation of chlorophylls and derivatives [8,84—86]. Studies of hydroporphyrins and chlorophylls also have been carried out with Raman resonance spectroscopy [87]. 

It was not possible to use MS for the characterization and identification of chlorophylls and its derivatives until the development of desorption methods (desorption ionization) appropriate for nonvolatile and thermolabile compounds. The mass spectmm of chlorophylls has been obtained using laser desorption [92-94], field desorption [95], plasma desorption [96,97], fast atom bombardment (FAB) [28, 98-101], in-beam electron ionization [102], and electrospray ionization (ESI) [103]. A combination of the techniques of desorption and tandem mass spectroscopy (MS/MS) has also been used for the charactmzation of chlorophylls and derivatives [ 104—107]. The latest research in this field coupled HPLC with MS, using as ionization source FAB [108-110] or atmosphoic pressure chemical ionization (APCI) [111-115]. 

Numerous works have been cited that use normal-phase silica columns and isocratic mixtures of solvents such as acetone/ligroin (20 80, v/v) for the quantification of chlorophylls and derivatives in phytoplankton [223], or iso-octane/98% ethanol (9 1, v/v) in spinach [224]. Watanabe et al. [225] separated chlorophylls and pheophytins using the isocratic mixture 2-propanol/n-hexane (3 97, v/v). This method yields chlorophylls with levels of purity above 99%. Abaychi and Riley [226], using the mixture petroleum ether/acetone/dimethylsulfoxide/diethylamine (75 23.25 1.5 0.25, v/v) as mobile phase, detected and quantified 16 pigments of chlorophylls, derivatives, and carotenoids. 

At the same time, nonaqueous reversed-phase methods have been developed. Wright and Shearer [220] used a linear gradient from 90% of acetonitrile to 100% of ethyl acetate to separate 44 pigments that included carotenes, xanthophylls, chlorophylls, and derivatives, in marine phytoplankton. Khachik et al. [240] combined an isocratic elution and gradient of methanol, acetonitrile, methylene chloride, and n-hexane to separate the major constituents (xanthophylls, chlorophylls, and caroteies) in different vegetables. 

Detection is normally by absorption or fluorescence spectrophotometry. The high coefficients of extinction of the chlorophyll Soret band enable sensitive detection between 380 and 445 nm. This region of the spectmm also includes the carotenoids, which accompany the chlorophylls in plant pigment extracts and whose analysis and quantification are also usually of interest (see Chapter 6). When these compounds are not of interest, and their possible interference must be excluded, a selective detection of chlorophylls and derivatives can be carried out at 654 [136] or 667 nm [230], where there is no absorption of these pigments. Detection... 

Table 7.4 shows the chromatographic and spectroscopic (electron absorption maxima in the eluent) characteristics obtained from the separation of chlorophylls and derivatives by HPLC and UV-Vis detection. Pigment identification is completed with assignation of IR spectrum bands and mass spectrum. 

Chromatographic Characteristics in Thin Layer Chromatography (TLC) of Chlorophylls and Derivatives... 

Chromatographic and Spectroscopic Characteristics in High-Performance Liquid Chromatography (HPLC) of Chlorophyll and Derivatives... 

Jones, I.D. et al.. An evaluation of reversed phase partition for thin-layer chromatographic identification of chlorophylls and derivatives, J. Chromatogr., 70, 87, 1972. 

Subsequent work by Zambiazi and Przybylski (1998) also showed that fatty acid composition could only explain half of the oxidative stability of vegetable oils including canola oil. The other half was attributed to the amount and composition of endogenous minor components which can shorten or extend the shelf-life of an oil. Such endogenous components were later discussed by Przybylski and Eskin (2006) and included tocopherols, mono- and diacylglycerols, free fatty acids, phospholipids, chlorophylls and derivatives, carotenoids, phytosterols, phenolic compounds and trace metals. In addition, the position that the fatty acid occupies in the triacylglyc-erol can also affect stability. For example, the location of linolenic and linoleic acids on the sn-2 position has been reported to cause faster oxidation and lower stability compared to the same fatty adds on ml- and sn-3 positions. In contrast oleic acid at the sn-2 position proved stabler compared to its location on sn-1 and sn-3 positions (Neffetal., 1994,1997). 

Tab. 1. Flurorescence life times of chlorophylls and derivatives (performed by Holzwarth, MPI Muhlheim/Ruhr.)...

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