Chromatographic Separation of Chlorophylls

F4.4 Chromatographic Separation of Chlorophylls F4.5 Mass Spectrometry of Chlorophylls... 

Schlotzhauer, W.S. Liquid chromatographic separation of chlorophyll products in tobacco leaf Tob. Sci. 22... 

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. 

In the post-World War II years, synthesis attained a different level of sophistication partly as a result of the confluence of five stimuli (1) the formulation of detailed electronic mechanisms for the fundamental organic reactions, (2) the introduction of conformational analysis of organic structures and transition states based on stereochemical principles, (3) the development of spectroscopic and other physical methods for structural analysis, (4) the use of chromatographic methods of analysis and separation, and (5) the discovery and application of new selective chemical reagents. As a result, the period 1945 to 1960 encompassed the synthesis of such complex molecules as vitamin A (O. Isler, 1949), cortisone (R. Woodward, R. Robinson, 1951), strychnine (R. Woodward, 1954), cedrol (G. Stork, 1955), morphine (M. Gates, 1956), reserpine (R. Woodward, 1956), penicillin V (J. Sheehan, 1957), colchicine (A. Eschenmoser, 1959), and chlorophyll (R. Woodward, 1960) (page 5). ... 

A normal-phase HPLC separation seems to be useful to separate major chlorophyll derivatives, but it is not compatible with samples in water-containing solvents an additional extraction step is required to eliminate water from the extract since its presence rednces chromatographic resolution and interferes with retention times. Besides that, the analysis cannot be considered quantitative due to the difhculty in transferring componnds from the acetone solution into the ether phase. On the other hand, an advantage of the normal-phase method is its efficacy to separate magne-sinm-chlorophyll chelates from other metal-chelated chlorophyll derivatives. ... 

In contrast to the well-established methods for identifying and quantifying naturally occurring chlorophylls, very few reports concern quantitative analysis of chlorophyllin copper complexes in color additives and in foodstuffs. Analytical methods proposed are based on spectral properties, elemental analysis, chromatographic separation, and molecular structure elucidation or a combination of these procedures. 

Hyvarinen, K. and Hynninen, P.H., Liquid chromatographic separation and mass spectrometric identification of chlorophyll b allomers, J. Ghromatogr. A, 837, 107, 1999. 

Alkaline hydrolysis (saponification) has been used to remove contaminating lipids from fat-rich samples (e.g., pahn oil) and hydrolyze chlorophyll (e.g., green vegetables) and carotenoid esters (e.g., fruits). Xanthophylls, both free and with different degrees of esterification with a mixture of different fatty acids, are typically found in fruits, and saponification allows easier chromatographic separation, identification, and quantification. For this reason, most methods for quantitative carotenoid analysis include a saponification step. 

For example, an alumina layer with a nonaqueons mobile phase was optimized for the separation of the taxoid fraction from ballast snbstances [5]. Figure 11.3 shows the densitogram obtained for Taxus baccata cmde extract chromatographed on the alnmina layer developed with nonaqueous elnents. The nse of ethyl acetate and dichloromethane enables elntion of nonpolar fractions (chlorophylls and waxes) and purification of the starting zone (Figure 11.3a). In this system, all taxoids are strongly retained on the alumina layer. The use of a more polar mobile phase... 

The use of appropriate analytical standards is important for successful chromatographic separation, identification, and quantification of chlorophyll derivatives. While chlorophyll a and b derivatives are readily available commercially (Sigma-Aldrich) both metal-free pheophytins and metalloporphyrin analogs such as Cu2+ and Zn2+ pheophytins are not. In most instances, these derivatives must be prepared from the parent Mg-chlorophyl standards prior to use. These simple synthesis techniques are based on the work of Schwartz (1984) and are to be utilized for the rapid and efficient preparation of metal-free, Cu2+ and Zn2+ pheophytin derivatives in quantities appropriate only for analytical implementation. 

Tests carried out with the high performance liquid chromatographic technique endorsed the claim that it causes negligible degradation of both the chlorophylls and the xanthophylls. A more efficient separation of... 

Several chromatographic methods have been published, and most of these utilise thin layer chromatography to separate the chlorophylls. Thin... 

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