Chlorophylls reversed-phase HPLC

The hrst HPLC method for chlorophyll analysis was developed by Evans and coworkers, in 1975 and since then a number of both normal and reversed phase HPLC methods have been employed. 

Basic Protocol Cl 8 Reversed-Phase HPLC Separation of Chlorophylls... 

C18 REVERSED-PHASE HPLC SEPARATION OF CHLOROPHYLLS a AND b AND THEIR NONPOLAR DERIVATIVES... 

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. 

RCl by using reverse-phase HPLC for separation and purification and concluded that Chl-RC 1 was a preparation artifact formed during the TLC separation step. The conclusion appeared reasonable as it is known that chlorophyll is easily chlorinated at the C20 position and readily hydroxylated on the silica-gel material used in thin-layer chromatography. 

For quantitation of cholesterol and its derivatives in muscle and liver tissues, the extracts of the tissue homogenates are evaporated, dissolved in a mobile phase such as hexane-isopropanol, and injected onto a normal-phase column. For analysis of soybean oil by reversed-phase HPLC, after extraction with chloroform-methanol (9 1), the neutral lipids, chlorophyls, and the phospholipids are separated by TLC. The lipids recovered from the TLC are analyzed by HPLC. 

Chlorophyll and carotenoid composition of LHCP from spinach and Chlorella were determined by reversed-phase HPLC (Braumann, Grimme,... 

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). 

The widespread application of HPLC methodology to chlorophyll analysis demonstrates its flexibility, effectiveness, and reliability. Methods described in this unit are based on the work of Schwartz et al. (1981). This original method allows for resolution of twelve chlorophyll derivatives in 30 min. While minor modifications were made to this method to further simplify the analysis, the final resolution and sensitivity were not compromised. Based on a commercially available reversed-phase column and an aqueous mobile phase, the method can be easily altered for specific separations. This is demonstrated in the Alternate Protocol, where the Basic Protocol was adjusted for the analysis of polar and Cu2+- and Zn2+-containing chlorophyll derivatives. The method for polar derivatives is based on the separation of Can-jura and Schwartz (1991), while the method for... 

C18 reversed-phase column for HPLC separations of chlorophylls narrow-bore (2.1 -mm i.d.) column at flow rate of 50 to 300 p.l/min without splitting the flow, or analytical column (4.6-mm i.d.) at 1 ml/min with post-column solvent splitting of 5 1 (200 p,l/min entering the mass spectrometer) for APCI electrospray interfaces are available for use without solvent splitting over all flow rates from nl/min to 1 ml/min... 

HPLC separation of the chlorophyll mixture should be carried out using reversed-phase (usually Cl8 columns) as described in UNIT F4.4. 

Zapata M, Rodriguez F, Garrido JL (2000) Separation of chlorophylls and carotenoids from marine phytoplanktons new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases. Mar Ecol Prog Ser 195 29-45... 

Figure 2 Reversed-phase LC separation of deuterated chlorophyll a and undeuterated chlorophyll a on a 25 cm x 4.6 mm i.d. Ci8-Ultrasphere CDS column. Mobile phase water-methanol-acetonitrile-tetrahydrofuran 5 28 38 23 (v/v/v/v). UV detector 663 nm. (Reprinted with permission from Baweja R, et al. (1986) HPLC separation of deuterated photosynthetic pigments from their protio analogues. Journal of Chromatography 369 125-131 Elsevier.)...

Other TLC studies on fat-soluble chloroplast pigments from plant tissues include the following. Suzuki et al. (1987) studied the retention of chlorophylls, pheophytins, and pheophorbides in C,g TLC and HPLC systems. Stauber and Jeffrey (1988) examined the photosynthetic pigments of 51 species of tropical and subtropical diatoms by normal- and reversed-phase TLC. Cserhati (1988) determined the lipophilicity of some photosynthetic pigments using reversed-phase TLC on various impregnated layers with acetone and ethanol as the mobile phase. Heimler et al. (1989) quantitatively determined chlorophylls in spruce needles by densitometry on cellulose layers. 

HPLC can also be used in the isolation of the different standard chlorophyll derivatives in their pure form, employing a semipreparative column of 25 cm in length and 1 cm internal diameter [28]. Each fraction collected in semipreparative HPLC is transferred to ethyl ether, and aqueous NaCl solution (10%) is added. The solvent is removed in a rotavapor, and each pure pigment fraction is subjected to different identification tests. This chromatographic development in reversed phase separates the pigments in direct relation to their polarity. The retention of a compound in HPLC is expressed by the capacity factor k, defined as... 

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