Analysis of chlorophyll

The most recent progress in MS analysis of chlorophylls has been obtained with the development of atmospheric ionization methods such as atmospheric pressure chemical ionization (APCl) and electrospray ionization (ESI). These techniques have demonstrated much more sensitivity than thermospray ionization, detecting chloro-... 

A. Cichelli and G.P. Pertesana, High-performance liquid chromatographic analysis of chlorophylls, pheophytins and carotenoids in virgin olive oils chemometric approach to variety classification. J. Chromatogr.A 1046 (2004) 141-146. 

Development of fast, accurate, and reproducible high-performance liquid chromatography (HPLC) methods has offset the use of traditional open-column and TLC methods in modern chlorophyll separation and analysis. A number of normal and reversed-phase methods have been developed for analysis of chlorophyll derivatives in food samples (unit F4.4), with octadecyl-bonded stationary phase (C]8) techniques predominating in the literature (Schwartz and Lorenzo, 1990). Inclusion of buffer salts such as ammonium acetate in the mobile phase is often useful, as this provides a proton equilibrium suitable for ionizable chlorophyllides and pheophorbides (Almela et al., 2000). 

This protocol focuses on the analysis of chlorophyll a and b, and the more nonpolar derivatives, including pheophytins and pyropheophytins. An octadecyl-bonded, reversed-phase stationary phase is used with a methanol/water mixture and ethyl acetate mobile phases in a gradient elution to provide rapid and complete separation of the major chlorophyll derivatives in 25 to 30 min. This is coupled with traditional UV/visible spectrophotometric detection at 654 nm to selectively screen these photosynthetic pigments in food and plant tissues. 

In widespread use since 1982 (Barber et al., 1982), FAB and LSIMS are matrix-mediated techniques. The most effective matrix for static FAB/LSIMS analysis of chlorophylls and their derivatives is 3-nitrobenzyl alcohol (van Bree-men et al., 1991a), whereas glycerol provides adequate sensitivity and a more robust system during continuous-flow FAB/LSIMS (van Breemen et al., 1991b). Ionization and desorption of the chlorophyll analyte occur together during the bombardment of the matrix by fast atoms (or ions) to produce molecular ions, M+-, and protonated molecules, [M+H]+. 

Both APCI and ESI are compatible with the same HPLC columns and solvent systems used for chlorophyll analysis. However, the main advantage of APCI compared to electrospray for chlorophyll analysis is the linearity of the detector response, which is more than two orders of magnitude larger for APCI. This suggests that APCI LC/MS might be preferred to ESI LC/MS for quantitative analysis of chlorophylls. 

D. Application HPLC Analysis of Chlorophylls, Chlorophyll Derivatives, and Carotenoids in Spinach (Ref. 99)... 

Wright SW, Jeffrey SW, Mantoura RFC, Llewellyn CA, Bjprnland T, Repeta D, Welschmeyer NA (1991) Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar Ecol Prog Ser 77 183-196... 

Welschmeyer, N. A., 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and pheopigments. Limnology and Oceanography, 39, 1985-1992. 

U. Schreiber, T. Endo, H. Mi, K. Asada (1995). Quenching analysis of chlorophyll fluorescence by the saturation pulse method Particular aspects relating to the study of eukaryotic algae and cyanobacteria. Plant Cell Physiol, 36, 873-882. 

The mild SDS-PAGE analysis of chlorophyll-protein complexes were performed as in(8). The grana and stroma lamellae were isolated as in(7). The analysis of polypeptide composition of grana and stroma lamellae and re-electrophoresis were performed according to (9). 

Electrophoresis. For analysis of chlorophyll-protein complexes (Table 2) a mild SDS-DAGE method (6) was used. Two dimensional gel electrophoresis (7) was used for analysis of 25 and 27 kDa polypeptides (Fig.l). 

The analysis of chlorophyll-protein complexes shows a drastic reduction in CP Ila and CP Ilb bands (associated with LHC II) in the thylakoids of MP treated seedlings (Plate.l). The reduction in LHC II could be either due to the direct interaction of MP v ith the synthesis of LPC II proteins or due to the failure in the assembly of the LHC II proteins. 

Two dimensional analysis of chlorophyll-proteins Light harvesting bands from deoxycholate green gels were incubated in denaturing solution (6), and separated according to Laemmli (7). 

FIGURE 2. Photosynthetic CO2 uptake, variable fluorescence kinetics and analysis of chlorophyll fluorescence quenching during changes of C02l in the gas-phase surronding a leaf fed with 1 mM PPT. 

Chlorophylls and their derivatives are foimd, for example, in many vegetables, and they are also used as food colorants. They have a strong hydrophobic character, so they are usually analyzed by RPLC with a C18 column and an organic mobile phase, except when chlorophyll c (polar molecule) is present. In this case, the addition of a small amount of water to the organic solvent is recommended. Spectrophotometric and MS detectors are the most commonly employed detection modes for the analysis of chlorophylls. 

For the analysis of chlorophylls and carotenoids, and their derivatives, in processed green plants, the previously described method can also be used. Figure 6.13 shows the chromatogram for the separation of chloroplast pigments of a processed green plant, green table olives, whose processing includes a fermentation step. 

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