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An improved method for the rapid and easy separation of leaf pigments and their derivatives by thin-layer chromatography
ANALYTICAL
BIOCHEMISTRY
106, 322-326 (1980)
An Improved Method for the Rapid and Easy Separation of Leaf Pigments and Their Derivatives by Thin-Layer Chromatography KEIJI
IRIYAMA, MASAHIKO SHINJI YANO,*
YOSHIURA, MASARU AND SHIRO SAITO*
SHIRAKI,
Research Insrirure for Polymers and Textiles, l-14 Yalabe-Higashi, Tsukuba, Ibaraki 305, Japan, and *Department of Electrical and Electric Engineering, Tokyo Insrirure of Technology, Meguro-ku, Tokyo 152, Japan Received January 2, 1980 A thin-layer chromatographic method for the separation and identification of leaf pigments and their degradation products on commercial silica gel layer has been developed to give a tool to examine the purity of chlorophyll preparations and the chemical stability of chlorophyll molecules during the course of the chlorophyll preparation. It has been confirmed that the developing solvent system (isopentane:tert-butyl alcohokacetone = 9055, v/v/v) is quite useful to separate the photosynthetic pigments and their degradation products which were commonly found during the course of in vitro chlorophyll studies.
Recently, a high-performance liquid chromatographic method for the quantitative analysis of the photosynthetic pigments and their degradation products has been developed to supplement spectrophotometric observations (1). In addition, Csorba et al. (2) have developed a method for the high-speed videodensitometric determination of chlorophyll and carotenes separated by thin-layer chromatography (tic)’ aiming to make it possible to analyze quantitatively and rapidly. On the other hand, the rapid and easy qualitative analysis of the leaf pigments and their degradation products, while not very time consuming, is still required to examine the purity of chlorophyll preparations as well as the chemical stability of chlorophyll molecules during the course of chlorophyll preparation, since it has been generally recognized that the possible contaminants in chlorophyll preparations are yellow leafpigments and degradation products of chlorophyll, and also that the spectroscopic determination of 1 Abbreviations used: tic, thin-layer chromatography; Chl, chlorophyll; Pheo, pheophytin; solvent system (I), isopentane: rert-butyl alcohol:acetone = 90: 55, v/v/v. 322
carotenoids and chlorophyll decomposition products in the presence of chlorophyll in the same extract is very difficult because of overlapping of their visible absorption bands. In our previous paper (3), a thin-layer chromatographic method for the separation of chlorophyll, its derivatives, and yellow plant pigments has been developed. However, for example, the separation of chlorophyll b’ (Chl b ‘) and yellow pigment could not be attained. In this article, we report on an improved thin-layer chromatographic method for the separation of leaf pigments and degradation products of chlorophyll which were commonly found during the course of in vitro chlorophyll studies as well as chlorophyll preparation. MATERIALS
AND METHODS
All the experiments in this study were performed at 20°C in total darkness or under dim green light unless otherwise stated. Acetone extract from spinach leaves was prepared according to the method of Iriyama et al. (4). Acetone extract was evaporated and dried in a vacuum desiccator and the 0003-2697/80/120322-05$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any fom reserved.
THIN-LAYER
CHROMATOGRAPHY
resultants thus obtained were dissolved in benzene to be examined by tic. Chlorophyll a (Chl a) and chlorophyll b (Chl b) were prepared from fresh spinach leaves according to the procedures of Iriyama et al. (45). Carotenes, lutein, violaxanthin, and neoxanthin used as standard pigments in determining unknown yellow pigments were also prepared from spinach leaves according to the thin-layer chromatographic method (3). Spectral characteristics of these yellow pigments were in good agreement with the literature values (6) within experimental error variation. Chlorophyll a’ (Chl a’), Chl b’, pheophytin a (Pheo a), pheophytin b (Pheo b), pheophytin a’ (Pheo a’), and pheophytin b’ (Pheo b’) were also cochromatographed as standard pigments of degradation products of chlorophylls for identifying unknown green pigments, as these degradation products of chlorophylls were commonly formed in leaf extract (e.g., acetone extract) or during the course of chlorophyll preparation as well as in the in vitro chlorophyll studies. Chl a’ and Chl b’ were prepared from Chl a and Chl b, respectively, according to the method recently developed (7). The method is based on the fact that chlorophyll molecules in completely dried pyridine solution are selectively converted to their epimers such as Chl a’ and Chl b’. Chl a (20 mg) was dissolved in 35 ml of pyridine and the solution was heated at 5060°C for 1 h in argon atmosphere. The solution heated was allowed to stand for 12 h at 20°C in argon atmosphere. The reaction mixture thus treated was evaporated and dried in a vacuum desiccator. The darkgreen resultant thus obtained was dissolved in 10 ml of 10% (v/v) diethyl ether in hexane. The solution was poured onto the sugar column (2.5 x 35 cm) and the pigments were developed by the solvent system (isopropyl alcohokhexane = 0.5:99.5, v/v). Chl u’ (3.9 mg) in’dry weight bases was obtained. Chl b’ was also prepared according to the same procedures used in preparing Chl a’.
OF LEAF
PIGMENTS
323
Pheo a, Pheo b, Pheo a’, and Pheo b’ were prepared from Chl u , Chl b, Chl a’, and Chl b’ , respectively, according to the method recently developed (7). As an example, the procedures for preparing Pheo a will be shown. A platinum plate (4 x 4 cm) pigmented with 10 mg of Chl a was immersed into 1 N HN03 for 3 h to convert Chl a to Pheo a and then the pigment layer was dissolved into 10 ml of diethyl ether. Thin-layer chromatographic analysis revealed that Chl a was converted to Pheo a without inducing any other degradation products of Chl a and also that visual spot of Chl a could not be detected on the thin-layer chromatogram thus treated. Further purification of Pheo a was accomplished by means of column chromatography with powdered sugar according to the same procedures in preparing Chl a and Chl b (4). Pheo a (9.1 mg) was obtained from 10 mg of Chl a. Pheo b, Pheo a’, and Pheo b’ were also prepared according to the same procedures described above. All the solvents and chemicals were of analytical reagent grade and were used without further purification unless otherwise mentioned. Commercial silica gel sheets, 5 x 10 cm (Wakogel plate, Wako Pure Chemical Ind. Ltd., Tokyo, Japan), were purchased and used. Solutions of test materials in benzene were spotted with a 2-~1 pipet 1.5 cm from the lower edge of the thin-layer sheet. Pigments spotted on the sheet were developed in thin-layer chambers. The atmosphere in the thin-layer chambers was preequilibrated with the developing solvents for at least 15 min before the sheets were inserted. After the solvent front had ascended 7 cm (about 15 min), the sheets were instantly dried by a flush of nitrogen gas and were photographed under ultraviolet light (365 nm). It was previously confirmed that no chemical degradations were observed for all the pigments at least under the present thinlayer chromatographic conditions.
324
IRIYAMA
ET AL.
RESULTS AND DISCUSSION
The thin-layer chromatogram of pigments in the freshly prepared acetone extract developed in the solvent system (tert-butyl alcohol:n-pentane = 10:90, v/v) is shown in Fig. la. Figures lb and c show the thin-layer chromatograms of Chl b’ and lutein, respectively, developed in the same solvent system. The spot of Chl b’ was completely overlapped with that of lutein on the silica gel sheet developed in the solvent system. The formation of Chl b’ disturbed the isolation of Chl a by means of column chromatography with powdered sugar, as the band of Chl6’ was apt to overlap with that of Chl a in the elution pattern. Indeed, the thin-layer chromatographic test revealed in some cases that Chl a preparation was contaminated with Chl b’ . In addition, when Chl b dissolved in 10% (v/v) aqueous acetone, the same solvent system used in extracting chlorophyll from spinach, was kept for 1 h, the thin-layer chromatographic analysis confirmed that Chl b was gradually transformed to Chl b ’ , Pheo b, and Pheo b’ (see Fig. Id). The separation between Pheo b and Pheo b’ spots has not been attained.
Front
carotenes =---+Pheo-b
Pheo-b
and
Pheo-b'
Chl-a Chl-b Chl-b Lutein Violaxanth
in
Neoxanthin
start
.--+-)(--*-*--*(a)
(b)
(c)
(d)
(e)
FIG. 1. Thin-layer chromatograms of (a) leaf pigments in the acetone extract freshly prepared, (b) Chl b’, (c) lutein, (d) Chl b and its degradation products, and (e) leaf pigments in the acetone extract. The chromatograms (a-d) were developed in the solvent system (rert-butyl alcohol:n-pentane = 10:90, v/v) and the chromatogram (e) was developed in the solvent system @err-butyl alcohol:benzene = 10:90, v/v). For the explanations of Chl a, Chl b , Pheo a, Pheo b, Pheo b’, and acetone extract, see the text.
Thus, the above developing solvent system is not useful to find the possible formation of Chl b’. Figure le shows the thin-layer chromatogram of pigments in the freshly prepared acetone extract developed in the
____-_____---____-.
Violaxanthin
FIG. 2. Thin-layer chromatograms of (a) leaf pigments in the acetone extract freshly prepared, (b) (Chl D + Chlb), (c) (Chla’ +Chlb’), (d) Pheo a, (e) Pheo b, (f) Pheo a’, (g) Pheo b’, (h) pheophytins (Pheo a, Pheo b, Pheo a’, and Pheo b’), and (i) mixed pigments (leaf pigments in the freshly prepared acetone extract, Chl a’, Chl b’, Pheo a, Pheo b, Pheo a’, and Pheo b’) developed in the solvent system (isopentane:tert-butyl alcohol:acetone = 9055, v/v/v). For the explanations of acetone extract, Chl a, Chl b, Chl a’, Chl b’, Pheo a, Pheo b, Pheo a’, and Pheo b’, see the text.
THIN-LAYER TABLE
CHROMATOGRAPHY
1
Rf VALUES OF LEAF PIGMENTS AND THEIR DECRADATION PRODWTS ON SILICA GEL SHEETS DEVELOPED IN THE SOLVENT SYSTEM (tert-BuTYL ALCOHOL: ACETONE:~SOPENTANE = 5590, v/v/v) R, value
Pigment Chlorophyll Chlorophyll Chlorophyll Chlorophyll Pheophytin Pheophytin Pheophytin Pheophytin Lutein Violaxanthin Neoxanthin
a a’ b
0.73 0.84 0.50
b'
0.64
a b a’ b’
0.86 0.84 0.86 0.85 0.40 0.16 0.06
solvent system (tert-butyl alcohol:benzene = 10:90, v/v). The spot of lutein was located between the spots of Chl b and violaxanthin, but the complete separation between Chl a and Chl b has not been attained. However, the solvent system may be useful to detect the possible presence of yellow pigments in the chlorophyll preparations. The thin-layer chromatogram of pigments in the freshly prepared acetone extract developed in the solvent system (I) (isopentane: terf-butyl alcohokacetone = 90:5:5, vl v/v) is shown in Fig. 2a. In addition, the thin-layer chromatograms of (Chl a + Chl b), (Chl a’ + Chl b’), Pheo a, Pheo b, Pheo a’, and Pheo b’ are shown in Figs. Zb-g, respectively. The Rr values of the pigments on the chromatograms are represented in Table 1. It was confirmed that the Rf values of Chl a, Chl b , Chl a ’ Chl b ‘, and pheophytins were considerably different from each other, enough to give clear separation among the pigment spots, although the Rf values of Pheo a, Pheo b, Pheo a’, and Pheo b’ were almost the same. Figure 2h shows the thinlayer chromatogram of the mixed pigments (Pheo a, Pheo b, Pheo a’, and Pheo b’). These pheophytins were gathered into one
OF LEAF PIGMENTS
325
spot on the chromatogram, but the effectiveness of the solvent system was not reduced in examining the purity of chlorophyll preparations and the chemical stability of chlorophyll molecules, because the spot of pheophytins was completely separated from the adjacent chromatographic spots (carotenes and Chl a’). Figure 2i shows the thin-layer chromatogram of the mixed pigments (pigments in the freshly prepared acetone extract + Chl a’ + Chl b’ + Pheo a + Pheo b + Pheo a’ + Pheo b’) developed in solvent system (I). Good separation among neoxanthin, violaxanthin, lutein, Chl b, Chl b’, Chl a, Chl a’, pheophytins which were gathered into one spot, and carotenes was attained. As described above, it is concluded that the solvent system (I) is quite useful to examine the purity of chlorophyll prepa-
FIG. 3. Relationship between the R,values of various kinds of pigments (leaf pigments and their degradation products) and the developing solvent compositions. The developing solvent compositions were obtained from the equation [l]/([l] + [2] x 100 (%), where [l] and [2] were milliliters of the solvent mixture (tert-butyl alcohol: acetone = l:l, v/v) and milliliters of isopentane, respectively.
326
IRIYAMA
rations and the chemical stability of chlorophyll molecules in vitro. It has been generally recognized that Rf values are strongly dependent on developing solvent systems and also that the resolution between spots is dependent on spot sizes as well as developing solvent systems. Figure 3 shows the relationship between the Rf values of leaf pigments and their degradation products and the developing solvent compositions. In the figure, the solvent composition was obtained from the equation [l]/([l] + [2]) x 100 (%), where [l] and [2] are milliliters of the solvent mixture (tert-butyl alcohohacetone = 1: 1, v/v) and milliliters of isopentane, respectively. The Rf values of the pigments examined should be widely distributed from 0 to 1 without any overlapping between adjacent spots to detect all the pigments contained in the test solution. In Fig. 3, it may be emphasized that the solvent system (I) used in Fig. 2 is an appropriate developing solvent. However, resolution of the pigment spot on a thin-layer chromatogram should be essentially dependent on the spot size, especially the spot width along the flow direction of developing solvent as well as the distance between the spot centers for two adjacent spots, 1 and 2. Generally, resolution in tic can be defined by the function R, = Adl W, where Ad is the distance between spot centers for adjacent chromatographic spots after separation and W is the average spot width (8). W is obtained by the equation W = ( W, + W,)/2, where W, and W, are the sizes of 1 and 2, respectively, along with the flow direction of developing solvent. For values of R, Z 1, the separation of the two spots is relatively complete (8). We have obtained the values for R, of the respective adjacent spots in the case of the thin-layer
ET AL.
chromatogram shown in Fig. 2i. The high values of R, (greater than 1) were attained for all the adjacent spots on the silica gel sheets developed in the solvent system. Solvent system (I) gave the maximum values of R, for several cases compared with the other developing solvent systems examined in this study. From these high values of R,, it has been reconfirmed that solvent system (I) is very useful in giving a complete separation among the leaf pigments and their degradation products except for separation among pheophytins. Fortunately, lack of separation among pheophytins on, the thin-layer chromatogram developed in the solvent system (I) did not disturb examination of the purity of chlorophyll preparations as well as the chemical stability of chlorophyll molecules in vitro, as the spots of Chl a and Chl b were always completely separated from the degradation products of chlorophylls and any other leaf pigments under the present thin-layer chromatographic conditions. REFERENCES I. Iriyama, K., Yoshiura, M., and Shiraki, M. (1978)J. Chromatogr. 154, 302-305. 2. Csorba, I., Buzas, Z., Polyak, B., and Bpross, L. (1979) J. Chromatogr. 172, 287-293. 3. Shiraki, M., Yoshiura, M., and Iriyama, K. (1978) Chem. Lett. (Tokyo), 103-104. 4. Iriyama, K., Shiraki, M., and Yoshiura, M. (1979) .I. Liquid Chromatogr. 2, 255-276. 5. Iriyama, K., Yoshiura, M., and Shiraki, M. (1979) J. Chem. Sot. D, 406-407. 6. Braumann, T., and Grimme, L. H. (1979) J. Chromatogr. 170, 264-268. 7. K. Iriyama, Yoshiura, M., and Amada, M. (1979) Bull. Res. Inst. Polym. Text., No. 121, 37-43, [in Japanese]. 8. Snyder, L. R. (1975) in Chromatography (Heftmann, E., ed.), pp. 46-76, Van Nostrand Reinhold, New York/Cincinnati/Toronto/London/Melbourne.
BIOCHEMISTRY
106, 322-326 (1980)
An Improved Method for the Rapid and Easy Separation of Leaf Pigments and Their Derivatives by Thin-Layer Chromatography KEIJI
IRIYAMA, MASAHIKO SHINJI YANO,*
YOSHIURA, MASARU AND SHIRO SAITO*
SHIRAKI,
Research Insrirure for Polymers and Textiles, l-14 Yalabe-Higashi, Tsukuba, Ibaraki 305, Japan, and *Department of Electrical and Electric Engineering, Tokyo Insrirure of Technology, Meguro-ku, Tokyo 152, Japan Received January 2, 1980 A thin-layer chromatographic method for the separation and identification of leaf pigments and their degradation products on commercial silica gel layer has been developed to give a tool to examine the purity of chlorophyll preparations and the chemical stability of chlorophyll molecules during the course of the chlorophyll preparation. It has been confirmed that the developing solvent system (isopentane:tert-butyl alcohokacetone = 9055, v/v/v) is quite useful to separate the photosynthetic pigments and their degradation products which were commonly found during the course of in vitro chlorophyll studies.
Recently, a high-performance liquid chromatographic method for the quantitative analysis of the photosynthetic pigments and their degradation products has been developed to supplement spectrophotometric observations (1). In addition, Csorba et al. (2) have developed a method for the high-speed videodensitometric determination of chlorophyll and carotenes separated by thin-layer chromatography (tic)’ aiming to make it possible to analyze quantitatively and rapidly. On the other hand, the rapid and easy qualitative analysis of the leaf pigments and their degradation products, while not very time consuming, is still required to examine the purity of chlorophyll preparations as well as the chemical stability of chlorophyll molecules during the course of chlorophyll preparation, since it has been generally recognized that the possible contaminants in chlorophyll preparations are yellow leafpigments and degradation products of chlorophyll, and also that the spectroscopic determination of 1 Abbreviations used: tic, thin-layer chromatography; Chl, chlorophyll; Pheo, pheophytin; solvent system (I), isopentane: rert-butyl alcohol:acetone = 90: 55, v/v/v. 322
carotenoids and chlorophyll decomposition products in the presence of chlorophyll in the same extract is very difficult because of overlapping of their visible absorption bands. In our previous paper (3), a thin-layer chromatographic method for the separation of chlorophyll, its derivatives, and yellow plant pigments has been developed. However, for example, the separation of chlorophyll b’ (Chl b ‘) and yellow pigment could not be attained. In this article, we report on an improved thin-layer chromatographic method for the separation of leaf pigments and degradation products of chlorophyll which were commonly found during the course of in vitro chlorophyll studies as well as chlorophyll preparation. MATERIALS
AND METHODS
All the experiments in this study were performed at 20°C in total darkness or under dim green light unless otherwise stated. Acetone extract from spinach leaves was prepared according to the method of Iriyama et al. (4). Acetone extract was evaporated and dried in a vacuum desiccator and the 0003-2697/80/120322-05$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any fom reserved.
THIN-LAYER
CHROMATOGRAPHY
resultants thus obtained were dissolved in benzene to be examined by tic. Chlorophyll a (Chl a) and chlorophyll b (Chl b) were prepared from fresh spinach leaves according to the procedures of Iriyama et al. (45). Carotenes, lutein, violaxanthin, and neoxanthin used as standard pigments in determining unknown yellow pigments were also prepared from spinach leaves according to the thin-layer chromatographic method (3). Spectral characteristics of these yellow pigments were in good agreement with the literature values (6) within experimental error variation. Chlorophyll a’ (Chl a’), Chl b’, pheophytin a (Pheo a), pheophytin b (Pheo b), pheophytin a’ (Pheo a’), and pheophytin b’ (Pheo b’) were also cochromatographed as standard pigments of degradation products of chlorophylls for identifying unknown green pigments, as these degradation products of chlorophylls were commonly formed in leaf extract (e.g., acetone extract) or during the course of chlorophyll preparation as well as in the in vitro chlorophyll studies. Chl a’ and Chl b’ were prepared from Chl a and Chl b, respectively, according to the method recently developed (7). The method is based on the fact that chlorophyll molecules in completely dried pyridine solution are selectively converted to their epimers such as Chl a’ and Chl b’. Chl a (20 mg) was dissolved in 35 ml of pyridine and the solution was heated at 5060°C for 1 h in argon atmosphere. The solution heated was allowed to stand for 12 h at 20°C in argon atmosphere. The reaction mixture thus treated was evaporated and dried in a vacuum desiccator. The darkgreen resultant thus obtained was dissolved in 10 ml of 10% (v/v) diethyl ether in hexane. The solution was poured onto the sugar column (2.5 x 35 cm) and the pigments were developed by the solvent system (isopropyl alcohokhexane = 0.5:99.5, v/v). Chl u’ (3.9 mg) in’dry weight bases was obtained. Chl b’ was also prepared according to the same procedures used in preparing Chl a’.
OF LEAF
PIGMENTS
323
Pheo a, Pheo b, Pheo a’, and Pheo b’ were prepared from Chl u , Chl b, Chl a’, and Chl b’ , respectively, according to the method recently developed (7). As an example, the procedures for preparing Pheo a will be shown. A platinum plate (4 x 4 cm) pigmented with 10 mg of Chl a was immersed into 1 N HN03 for 3 h to convert Chl a to Pheo a and then the pigment layer was dissolved into 10 ml of diethyl ether. Thin-layer chromatographic analysis revealed that Chl a was converted to Pheo a without inducing any other degradation products of Chl a and also that visual spot of Chl a could not be detected on the thin-layer chromatogram thus treated. Further purification of Pheo a was accomplished by means of column chromatography with powdered sugar according to the same procedures in preparing Chl a and Chl b (4). Pheo a (9.1 mg) was obtained from 10 mg of Chl a. Pheo b, Pheo a’, and Pheo b’ were also prepared according to the same procedures described above. All the solvents and chemicals were of analytical reagent grade and were used without further purification unless otherwise mentioned. Commercial silica gel sheets, 5 x 10 cm (Wakogel plate, Wako Pure Chemical Ind. Ltd., Tokyo, Japan), were purchased and used. Solutions of test materials in benzene were spotted with a 2-~1 pipet 1.5 cm from the lower edge of the thin-layer sheet. Pigments spotted on the sheet were developed in thin-layer chambers. The atmosphere in the thin-layer chambers was preequilibrated with the developing solvents for at least 15 min before the sheets were inserted. After the solvent front had ascended 7 cm (about 15 min), the sheets were instantly dried by a flush of nitrogen gas and were photographed under ultraviolet light (365 nm). It was previously confirmed that no chemical degradations were observed for all the pigments at least under the present thinlayer chromatographic conditions.
324
IRIYAMA
ET AL.
RESULTS AND DISCUSSION
The thin-layer chromatogram of pigments in the freshly prepared acetone extract developed in the solvent system (tert-butyl alcohol:n-pentane = 10:90, v/v) is shown in Fig. la. Figures lb and c show the thin-layer chromatograms of Chl b’ and lutein, respectively, developed in the same solvent system. The spot of Chl b’ was completely overlapped with that of lutein on the silica gel sheet developed in the solvent system. The formation of Chl b’ disturbed the isolation of Chl a by means of column chromatography with powdered sugar, as the band of Chl6’ was apt to overlap with that of Chl a in the elution pattern. Indeed, the thin-layer chromatographic test revealed in some cases that Chl a preparation was contaminated with Chl b’ . In addition, when Chl b dissolved in 10% (v/v) aqueous acetone, the same solvent system used in extracting chlorophyll from spinach, was kept for 1 h, the thin-layer chromatographic analysis confirmed that Chl b was gradually transformed to Chl b ’ , Pheo b, and Pheo b’ (see Fig. Id). The separation between Pheo b and Pheo b’ spots has not been attained.
Front
carotenes =---+Pheo-b
Pheo-b
and
Pheo-b'
Chl-a Chl-b Chl-b Lutein Violaxanth
in
Neoxanthin
start
.--+-)(--*-*--*(a)
(b)
(c)
(d)
(e)
FIG. 1. Thin-layer chromatograms of (a) leaf pigments in the acetone extract freshly prepared, (b) Chl b’, (c) lutein, (d) Chl b and its degradation products, and (e) leaf pigments in the acetone extract. The chromatograms (a-d) were developed in the solvent system (rert-butyl alcohol:n-pentane = 10:90, v/v) and the chromatogram (e) was developed in the solvent system @err-butyl alcohol:benzene = 10:90, v/v). For the explanations of Chl a, Chl b , Pheo a, Pheo b, Pheo b’, and acetone extract, see the text.
Thus, the above developing solvent system is not useful to find the possible formation of Chl b’. Figure le shows the thin-layer chromatogram of pigments in the freshly prepared acetone extract developed in the
____-_____---____-.
Violaxanthin
FIG. 2. Thin-layer chromatograms of (a) leaf pigments in the acetone extract freshly prepared, (b) (Chl D + Chlb), (c) (Chla’ +Chlb’), (d) Pheo a, (e) Pheo b, (f) Pheo a’, (g) Pheo b’, (h) pheophytins (Pheo a, Pheo b, Pheo a’, and Pheo b’), and (i) mixed pigments (leaf pigments in the freshly prepared acetone extract, Chl a’, Chl b’, Pheo a, Pheo b, Pheo a’, and Pheo b’) developed in the solvent system (isopentane:tert-butyl alcohol:acetone = 9055, v/v/v). For the explanations of acetone extract, Chl a, Chl b, Chl a’, Chl b’, Pheo a, Pheo b, Pheo a’, and Pheo b’, see the text.
THIN-LAYER TABLE
CHROMATOGRAPHY
1
Rf VALUES OF LEAF PIGMENTS AND THEIR DECRADATION PRODWTS ON SILICA GEL SHEETS DEVELOPED IN THE SOLVENT SYSTEM (tert-BuTYL ALCOHOL: ACETONE:~SOPENTANE = 5590, v/v/v) R, value
Pigment Chlorophyll Chlorophyll Chlorophyll Chlorophyll Pheophytin Pheophytin Pheophytin Pheophytin Lutein Violaxanthin Neoxanthin
a a’ b
0.73 0.84 0.50
b'
0.64
a b a’ b’
0.86 0.84 0.86 0.85 0.40 0.16 0.06
solvent system (tert-butyl alcohol:benzene = 10:90, v/v). The spot of lutein was located between the spots of Chl b and violaxanthin, but the complete separation between Chl a and Chl b has not been attained. However, the solvent system may be useful to detect the possible presence of yellow pigments in the chlorophyll preparations. The thin-layer chromatogram of pigments in the freshly prepared acetone extract developed in the solvent system (I) (isopentane: terf-butyl alcohokacetone = 90:5:5, vl v/v) is shown in Fig. 2a. In addition, the thin-layer chromatograms of (Chl a + Chl b), (Chl a’ + Chl b’), Pheo a, Pheo b, Pheo a’, and Pheo b’ are shown in Figs. Zb-g, respectively. The Rr values of the pigments on the chromatograms are represented in Table 1. It was confirmed that the Rf values of Chl a, Chl b , Chl a ’ Chl b ‘, and pheophytins were considerably different from each other, enough to give clear separation among the pigment spots, although the Rf values of Pheo a, Pheo b, Pheo a’, and Pheo b’ were almost the same. Figure 2h shows the thinlayer chromatogram of the mixed pigments (Pheo a, Pheo b, Pheo a’, and Pheo b’). These pheophytins were gathered into one
OF LEAF PIGMENTS
325
spot on the chromatogram, but the effectiveness of the solvent system was not reduced in examining the purity of chlorophyll preparations and the chemical stability of chlorophyll molecules, because the spot of pheophytins was completely separated from the adjacent chromatographic spots (carotenes and Chl a’). Figure 2i shows the thin-layer chromatogram of the mixed pigments (pigments in the freshly prepared acetone extract + Chl a’ + Chl b’ + Pheo a + Pheo b + Pheo a’ + Pheo b’) developed in solvent system (I). Good separation among neoxanthin, violaxanthin, lutein, Chl b, Chl b’, Chl a, Chl a’, pheophytins which were gathered into one spot, and carotenes was attained. As described above, it is concluded that the solvent system (I) is quite useful to examine the purity of chlorophyll prepa-
FIG. 3. Relationship between the R,values of various kinds of pigments (leaf pigments and their degradation products) and the developing solvent compositions. The developing solvent compositions were obtained from the equation [l]/([l] + [2] x 100 (%), where [l] and [2] were milliliters of the solvent mixture (tert-butyl alcohol: acetone = l:l, v/v) and milliliters of isopentane, respectively.
326
IRIYAMA
rations and the chemical stability of chlorophyll molecules in vitro. It has been generally recognized that Rf values are strongly dependent on developing solvent systems and also that the resolution between spots is dependent on spot sizes as well as developing solvent systems. Figure 3 shows the relationship between the Rf values of leaf pigments and their degradation products and the developing solvent compositions. In the figure, the solvent composition was obtained from the equation [l]/([l] + [2]) x 100 (%), where [l] and [2] are milliliters of the solvent mixture (tert-butyl alcohohacetone = 1: 1, v/v) and milliliters of isopentane, respectively. The Rf values of the pigments examined should be widely distributed from 0 to 1 without any overlapping between adjacent spots to detect all the pigments contained in the test solution. In Fig. 3, it may be emphasized that the solvent system (I) used in Fig. 2 is an appropriate developing solvent. However, resolution of the pigment spot on a thin-layer chromatogram should be essentially dependent on the spot size, especially the spot width along the flow direction of developing solvent as well as the distance between the spot centers for two adjacent spots, 1 and 2. Generally, resolution in tic can be defined by the function R, = Adl W, where Ad is the distance between spot centers for adjacent chromatographic spots after separation and W is the average spot width (8). W is obtained by the equation W = ( W, + W,)/2, where W, and W, are the sizes of 1 and 2, respectively, along with the flow direction of developing solvent. For values of R, Z 1, the separation of the two spots is relatively complete (8). We have obtained the values for R, of the respective adjacent spots in the case of the thin-layer
ET AL.
chromatogram shown in Fig. 2i. The high values of R, (greater than 1) were attained for all the adjacent spots on the silica gel sheets developed in the solvent system. Solvent system (I) gave the maximum values of R, for several cases compared with the other developing solvent systems examined in this study. From these high values of R,, it has been reconfirmed that solvent system (I) is very useful in giving a complete separation among the leaf pigments and their degradation products except for separation among pheophytins. Fortunately, lack of separation among pheophytins on, the thin-layer chromatogram developed in the solvent system (I) did not disturb examination of the purity of chlorophyll preparations as well as the chemical stability of chlorophyll molecules in vitro, as the spots of Chl a and Chl b were always completely separated from the degradation products of chlorophylls and any other leaf pigments under the present thin-layer chromatographic conditions. REFERENCES I. Iriyama, K., Yoshiura, M., and Shiraki, M. (1978)J. Chromatogr. 154, 302-305. 2. Csorba, I., Buzas, Z., Polyak, B., and Bpross, L. (1979) J. Chromatogr. 172, 287-293. 3. Shiraki, M., Yoshiura, M., and Iriyama, K. (1978) Chem. Lett. (Tokyo), 103-104. 4. Iriyama, K., Shiraki, M., and Yoshiura, M. (1979) .I. Liquid Chromatogr. 2, 255-276. 5. Iriyama, K., Yoshiura, M., and Shiraki, M. (1979) J. Chem. Sot. D, 406-407. 6. Braumann, T., and Grimme, L. H. (1979) J. Chromatogr. 170, 264-268. 7. K. Iriyama, Yoshiura, M., and Amada, M. (1979) Bull. Res. Inst. Polym. Text., No. 121, 37-43, [in Japanese]. 8. Snyder, L. R. (1975) in Chromatography (Heftmann, E., ed.), pp. 46-76, Van Nostrand Reinhold, New York/Cincinnati/Toronto/London/Melbourne.