Chlorophyll-a-epimer

Fig. 2.35. HPLC absorbance chromatograms of cultures of Phaeocyctis globosa colonies and Imantonia rotunda made at 436 nm. Retention time is given in minutes. AU = absorption units. Pigments 1, chlorophyll-c 2, chlorophyll-Cj+c2 3, cw-flucoxanthin 4, 19 -butanoxyloxyfucoxanthin 5, fucoxan-thin 6, 19 -hexanoxyloxyfucoxanthin 7, diadinoxanthin 8, diatoxanthin 9, phytilated chlorophyll-elite 10, chlorophyll-a allomer 11, chlorophyll-a 12, chlorophyll-a epimer 13, / ,/ -carotene. Reprinted with permission from E. Antajan et al. [78].

Fig. 2.128. HPLC-MS summed base peak mass chromatograms of total extracts of (a) T. weissflogii culture, (b) control, (c) faecal pellets immediately after grazing (48h) and (d) pellets after ageing in filtered seawater in the dark for 30 days. Peak identification a = phaeophorbide-a b = pyrophaeophorbide-a c = 132-chlorophyllone-a d = 132-epi-chlorophyllone-a (carotenoid) e + f = 132-hydroxyhlorophyll-a, 15 -hydroxylactone, chlorophyll-a g = chlorophyll-a g = chlorophyll-a epimer h = chlorophyll-a-like i = hydroxyphaeophytin-a + unknown i = hydroxyphaeophytin-a epimer j = phaeophytin-a j = phaeophytin-a epimer k = purpurin-18-phytyl ester 1 = pyrophaeophytin-a m = chlorine-a-like t = 132-oxopyrophaeophytin-a u = 132-oxopyrophaeophorbide a-24-methylcholesta-5,24(28)-dien-3/ -yl-ester SCE Sterol n = C272 d.b.a o = C27 2 d.b. +C28 2 d.b. p = C29 2 d.b. q = C27 1 d.b. r = C28 1 d.b. + C29 2 d.b. s = C29 2 d.b. Reprinted with permission from H. M. Talbot et al. [293], (ad.b = number of double bonds. = carotenoid.)...

Fig. 5. (A) Structure of the chlorophyll-a epimer (B) The in vitro oxidized-minus-reduced difference spectrum of dimeric Chi a compared with that of P700 (of spinach). Figure source Watanabe, Kobayashi, Hongu, Nakazato, Hiyama and Murata (1985) Evidence that a chlorophyll a dimer constitutes the photochemical reaction centre (P700) in photosynthetic apparatus. FEBS Lett 191 2255. The light-induced difference spectrum for P700 was originally in Hiyama and Ke (1972) Difference spectra and extinction coefficient of P700. Biochim Biophys Acta 267 163.

Rontani et al. [1266] studied the photodegiadation of chlorophylls using a Cjg column (A = 475nm) and a 15-min 90/10->50/50 acetonitrile/IPA gradient. Fucoxanthin, diadinoxanthin, chlorophyll a allomer, chlorophyll a and chlorophyll a epimer were well resolved. Peak shapes were reasonable and elution was complete in 12 min. 

The typical isocyclic ring E present in chlorophylls is susceptible to a number of different modifications such as epimerization, which produces stereoisomers by inversion of the configuration at C-13 of their parent pigments. These 13 -epichlorophylls, known as chlorophylls a and b, are minor pigments. They are considered artifacts produced in the course of handling plant extracts and sometimes are also found in small amounts in heated and deep-frozen vegetables, hi the old Fischer systan of nomenclature that can still be found in some literature, these epimers were named 10-epichlorophylls. 

Katz, J.J., Norman, G.D., Svec, W.A., and Strain, H.H. 1968. Chlorophyll diastereoisomers. The nature of chlorophylls a and b and evidence for bacteriochlorophyll epimers from proton magnetic resonance studies. J. Am. Chem. Soc. 90 6841-6848. 

Fig. 6. (A) Effect of composition of a binary soivent mixture on the apparent Chi a Aotal chlorophyli moiar ratio in extracts of spinach leaf tissue (0) and chloroplasts ( ). (B) relationship between the Chi a/Chl a molar ratio and the Chi a/P700 molar ratio for a number of P700-enriched subchloroplast particles by chloroform extraction (o) and by acetone extraction ( ). The solid line ciosest to the open circles is for Chi aVP700=1 and that nearest the filled circles for Chi aVP700= 2. See text for details. Figure source (A) Watanabe, Kobayashi, Maeda, Oba, Yoshida, Van de Meent and Amesz (1992) Function of the C13 -epimer chlorophylls in type I photosystem reaction centers. In N Murata (ed) Research In Photosynthesis, Vol III 4. Kluwer Acad PubI (B) Maeda, Watanabe, Kobayashi and Ikegami (1992) Presence of two chlorophyll a molecules at the core of photosystem I. Biochim Biophys Acta 1099 78.

FIGURE 7.12 High-performance liquid chromatography (HPLC) separation of pigments present in a green tissue. Peaks 1, neoxanthin 1, neoxanthin isomer 2, violaxanthin 2, violaxanthin isomer 3, luteoxanthin 4, anteraxanthin 5, lutein 5 and 5", lutein isomers 6, chlorophyll b 6, chlorophyll b C-132 epimer 7, chlorophyll a 7, chlorophyll a C-13 epimer 8, (i-carotene 8, cw-P-carotene isomer. 

Kostiainen, R., Hyvarinen, K., and Hynninen, P.H., Fast-atom bombardment mass spectra of the 132-epimers of 132-chlorophyll a. Rapid Comma. Mass Spectrom., 9, 555, 1995. 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

Usually, HPLC analysis resolves four peaks identified by co-chromatography with authentic standards as copper pheophorbide a, Cn(II) chlorin e6, Cn(II) chlorin e4, Cu rhodin g7, and their degradation products, but a sum of other colored components can also be found, for example, native chlorophylls, pheophytins, pheophor-bides, and rodochlorins (free carboxyl forms of pheophorbides) besides epimers, allomers, and degradation products that have been only tentatively identified. 

Scheme 5. In higher plants pheophoibide a (5a) is degraded to the primary fluorescent chlorophyll catabolite (pFCC, 10) and to its C(l)-epimer epi-10 (epi-pFCC)...

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