Chlorophyll quantification analysis

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. 

The detection and quantification of tocopherols, carotenoids, and chlorophylls in vegetable oil were effectively used for authentication pnrposes. The presence of tocopherols, carotenoids, and chlorophylls influence the oxidative stability of vegetable oils and their potential health benefits. Puspitasari-Nienaber et demonstrated the application of a rapid and reliable analysis method of direct injection of C-30 RP-NPLC with electrochemical detection for the simultaneous analysis of the above mentioned substances. Aliquots of vegetable oils were dissolved in appropriate solvents and injected directly without saponification, thus preventing sample loss or component degradation. Thus the effective separation of tocopherols, carotenoids, and chlorophylls was achieved. 

Saponification is the technique most used for the removal of fatty matter and other components such as chlorophylls (when their analysis is not required). In addition, saponification hydrolyzes the fatty acid esters of xanthophylls present in many ripe fruits, facilitating subsequent stages of analysis (such as isolation, identification, and quantification). The general procedure of pigment extract saponification is usually preceded by a step of transfer to diethyl ether, which is immiscible with water and has a low boiling point (below 35°C), simplifying water removal and its own removal by evaporation. This transfer not only helps saponification, but also prevents the formation of saponification artifacts, above all by reaction between ketones (usually because of the presence of acetone in the extract) and apocarotenal aldehyde groups. 

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... 

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