Chlorophyll, Separation

For convenience of discussion, a schematic diagram of bacterial photosynthetic RC is shown in Fig. 1 [29]. Conventionally, P is used to represent the special pair, which consists of two bacterial chlorophylls separated by 3 A, and B and H are used to denote the bacteriochlorophyll and bacteriopheophytin, respectively. The RC is embedded in a protein environment that comprise L and M branches. The initial electron transfer (ET) usually occurs from P to Hl along the L branch in 1—4 picoseconds (ps) and exhibits the inverse temperature dependence that is, the lower the temperature, the faster the ET. It should be noted that the distance between P and Hl is about 15 A [53-55]. 

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). 

Chlorophyll b [519-62-0] M 907.52, sinters at 86-92 , sinters at 170 , dec at 160-170 , m 183-185 , 190-195 , [alj, -267 (Me2CO + McOH), [a] j-133 (McOH + Pyridine 95 5). See purification of chlorophyll a, and is separated from "a" by chromatography on sucrose [UV, IR Stoll and Weidemann Helv Chim Acta 42 679, 681 7959]. It forms red-black hexagonal bipyramids or four sided plates from dilute EtOH and has been recrystd from CHCl3-MeOH. It is soluble in MeOH, EtOH, EtOAc and insoluble in pet ether. [J Am Chem Soc 88 5037 1966.]... 

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). ... 

Chlorophyll, as extracted from plants, is actually made up of two closely related compounds, chlorophyll A and chlorophyll B. These differ slightly in molecular structure and can be separated because they have different tendencies to be adsorbed on a finely divided solid (such as powdered sugar). 

Khachik, R, Beecher, G.R., and Whittaker, N.R, Separation, identification and quantification of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid chromatography, J. Agric. Food Chem., 34, 603, 1986. 

The methods for extraction and separation of chlorophylls and their derivatives and the most common procedures of identification and quantification are described in this chapter. 

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. 

Because plants present chlorophylls and carotenoids simultaneously, it may be useful to separate both groups from each other in a laboratory or preparative scale in order to avoid contamination in further purification steps, mainly when they are prepared in large amounts. Clean-up procedures using an open column packed with absorbents such as alumina, magnesia, polyethylene powder, powdered sucrose, DEAE-Sepharose, starch, cellulose, or MgO HyfloSupercel are good approaches. MgO HyfloSupercel in a proportion of 1 1 or 1 2 is the usual adsorbent. Sucrose and cellulose are interesting as they do not alter the chlorophylls, but they are tedious to work with. 

The most common mobile phase is a gradient of petroleum ether or hexane with increasing concentrations of acetone or diethyl ether. Development of the column should be optimized for each sample to afford a quick and effective separation to avoid band broadening. The separation can be followed visually. The most non-polar a- and 3-carotenes are eluted first as a yellow band followed by the chlorophylls and other more polar carotenoids like cryptoxanthin, lutein, and zeaxanthin that frequently fuse together and appear as a single band. ... 

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. ... 

Many other authors, as reviewed extensively by Schwartz and Lorenzo, and by Eder improved the C18 RP-HPLC methods that have been largely applied using similar but not exactly identical systems to separate and to quantify complex mixtures of chlorophylls and carotenoids. 

However, since fluorometric methods require sophisticated instrumentation, their applicabihty is limited because of cost. In conclusion, spectroscopic methods usually enable crude estimates of chlorophylls in an extract, but in most cases accurate and detailed analysis of a specific composition requires separation of the mixture into individual compounds using methods such as HPLC. 

All the analytical methods mentioned to separate, identify, and quantify chlorophylls and derivatives consume time, money, and samples. As alternatives, industries have been employing non-destructive methods for surface color measurements that are not only indirectly related to chlorophyll content, but may also estimate the pigments directly in tissues, leaving the sample intact and enabling serial analyses in a relatively short time. Eood color affects consumer acceptance and is an important criterion for quality control. Color vision is a complex phenomenon that depends on both the total content and number of pigments and also on absorption, reflectance and emission spectra of each compound present. 

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. 

Canjura, F.L. and Schwartz, S.J., Separation of chlorophyll compounds and then-polar derivatives by high-performance liquid chromatography, J. Agric. Food Ghem., 39, 1102, 1991. 

Shioi, Y, Doi, M., and Sasa, T., Separation of non-esterified chlorophylls by ion-suppression high-performance liquid chromatography, J. Ghromatogr., 298, 141, 1984. 

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

Buskov, S., Sprensen, H., and Sprensen, S., Separation of chlorophylls and their degradation products using packed column supercritical fluid chromatography (SEC), J. High Resol. Chromatogr, 22, 339, 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. 

Typically lipids, chlorophyll, and phenolic acids can be separated by liquid-liquid partition. Lipids and chlorophyll can be removed from acetone-water extracts by chloroform while phenolic acids have higher affinities for ethyl acetate at a pH close to nentral and water. °°... 

As plant extracts mainly comprise large amonnts of ballast substances (e.g., lipids and chlorophylls), their purification is often a priority in the analysis. Such purification can be expensive in terms of both time and solvent consumed and can lead to losses of sample components. Online purification and separation of extracts contaminated with plant oil, can be readily performed by TLC in equilibrium chambers [1] that enable the use of continuous elution. 

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