Chromatograms, pheophytin

Fig. 2.132. Chromatogram of spinach, stored frozen until analysis by HPLC (A) and after acidifying the same pigment extract with 0.2ml M HC1 per 10 ml extract and exposure to air and light for 15 h at 20°C (B). Zinc-phtalocyanine was used as an internal standard (IS). Peak identification 1 = chlorophyll-b 2 = chlorophyll-a x = unknown degradation product 3 = IS 4 = pheophytin-b 5 = pheophytin-a 6 = chlorophyll-b 7 = chlorophyll-a 8 = pheophytin-b 9 = pheophytin-a. Reprinted with permission from T. Bohn et al. [303].

Fig. 2.133. HPLC chromatogram of pigment extracts from table olives cv. Gordal (a) healthy fruits and (b) altered fruits. Peaks 1 = 15-glyoxilic acid pheophorbide-b 2 = 15-glyoxilic acid pheophorbide-a 3 = Cu-15-glyoxilic acid pheophorbide-a 4 = pheophorbide-b 5 = pheophorbide-a 6 = pyropheophorbide-a 7 = 15-glyoxilic acid pheophytin-b 8 = Cu-15-glyoxilic acid pheophytin-b 9 = 15-glyoxilic acid pheophytin-a 10 = Cu-15-glyoxilic acid pheophytin-a 11 = 15 -OH-lactone-pheophytin-b 12 = 15 -OH-lactone-pheophytin-a 13 = 15-formylpheophytin-b 14 = pheophytin-b 14 = pheophytin-b 15 = 15-formylpheophytin-a 16 = pheophytin-a 16 = pheophytin-a 17 = pyropheophytin-b 18 = Cu-pheophytin-a 19 = Cu-15-formylpheophytin-a 20 = pyropheophytin-a 21 = Cu-pyropheophytin-a. Reprinted with permission from B. Ganul-Rojas el al. [304].

Figure F4.4.1 Typical HPLC chromatogram of eight major chlorophyll derivatives separated using the Basic Protocol. Peak identifications 1, chlorophyll tr, 2, chlorophyll ti 3, chlorophyll a 4, chlorophyll a 5, pheophytin b 6, pyropheophytin b, 7, pheophytin a 8, pyropheophytin a.

Polar chlorophyll derivatives and metalloporphyrin derivatives such as Cu2+ and Zn2+ pheophytins can also be analyzed by C18 reversed-phase HPLC. Appropriate standards must be used see UNITF4.2 for polar chlorophyll derivatives, or see Support Protocol 2 for Cu2+ and Zn2+ pheophytin standards. Gradient solvent conditions and flow rates are given in Tables F4.4.3 and F4.4.4. Otherwise, the separation is performed as described for chlorophylls and nonpolar derivatives (see Basic Protocol). Using this method, separation of polar chlorophyll derivatives can be achieved in 20 to 25 min, and separation of the metalloporphyrin derivatives in 20 to 25 min. Examples of chromatograms obtained for polar derivatives, Zn2+ pheophytins, and Cu2+ pheophytins are shown in Figures F4.4.2, F4.4.3, and F4.4.4, respectively. 

Figure F4.4.2 HPLC chromatogram of chlorophyll derivatives separated using the Alternate Protocol. Peak identifications 1, chlorophyllide if 2, chlorophyllide a 2, chlorophyllide a" 3, pheophorbide tr, 3, pheophorbide if 4, pyropheophorbide b 5, pheophorbide a 5, pheophorbide a 6, pyropheophorbide a 7, chlorophyll tr, 7, chlorophyll if 8, chlorophyll a 8, chlorophyll a 9, pheophytin tr, 9, pheophytin tf 10, pyropheophytin tr, 11, pheophytin a 11, pheophytin a 12, pyropheophytin a. Reproduced from Canjura et al. (1991) with permission from the Institute of Food Technologists.

Figure F4.4.3 HPLC chromatogram of Zn2+ pheophytins separated using the Alternate Protocol. Peak identifications A, allomerized Zn2+ pheophytin b B, Zn2+ pheophytin b C, allomerized Zn2+ pheophytin a D, Zn2+ pheophytin a. Reproduced from Schwartz (1984) with permission from Marcel Dekker, Inc.

Fig. 9 Reversed-phase chromatogram of frozen pea extract. The C, 8-column was subjected to an isocratic elution by acetone water (70 17 13). The chlorophylls and pheophytins were detected by fluorescence, with the excitation wavelength at 413 nm and the emission wavelength at 669 nm. (From Ref. 107.)...

Fig. 11 Chromatogram of chlorophylls, chlorophyll derivatives, and carotenoids extracted from spinach leaves and analyzed by HPLC with a photodiode array detector. Chd = chlorophyllide, Pb = pheophorbide, Chi = chlorophyll, Po = pheophytin. (From Ref. 99.)...

The chromatogram obtained on separating pheophytins a and b from their respective Cu and Zn complexes (Figure 7.19) [27] enables detection of a possible alteration or adulteration of food color. As can be deduced from the retention times of these complexes, their separation from the rest of the pigments is perfectly practicable. 

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