- Email: [email protected]
Polyethylene-based high-performance liquid chromatography of chloroplast pigments: Resolution of mono- and divinyl chlorophyllides and other pigment mixtures
ANALYTICAL
BIOCHEMISTRY
162,493-499
(1987)
Polyethylene-Based High-Performance Liquid Chromatography Chloroplast Pigments: Resolution of Mono- and Divinyl Chlorophyllides and Other Pigment Mixtures’ Yuzo Division
of Biology,
SHIOI’
AND SAMUEL
Miyazaki Medical College, Kiyotake, Miyazaki and Medicine, Brown University, Providence,
of
I. BEALE* 889-16, Japan, and *Division Rhode Island 02912
of Biology
Received October 3 1, 1986 In addition to most chlorophylls and their derivatives, monovinyl and divinyl chlorophyll species were separated by high-performance liquid chromatography, using a polyethylene column and a simple elution with aqueous acetone. Peak retention and resolution of the pigment separation were greatly increased by increasing the polarity of the mobile phase and also by decreasing the column temperature. Polyethylene chromatography showed chlorophyll separation behavior similar to that of the octadecyl silica column, but it showed no adsorption of the pigment species containing free carboxylic acid groups, enabling the complete separation of chlorophylls and their derivatives. Polyethylene is a superior alternative stationary phase to the known reversed-phase materials for chlorophyll separation and analysis. o 1987 Academic press ITIC. KEY WORDS: chlorophyll separation; HPLC; mono- and divinyl chlorophylls; HPLC, polyethylene column.
Recently, it has become apparent that chlorophyll (Chl)3-pigment pools of etiolated and greening plants are not chemically homogeneous and that these pigment pools consist of MV and DV components (1). The detection of both MV and DV forms of Pchlide has been accomplished by fluorescence spectroscopy (2), but separation techniques for these compounds have not been fully developed. HPLC is commonly used for the separation and analysis of plant and bacterial pigments (3- 13). We have used this technique ’ Part of this work was done in Japan. 2 Recipient of a fellowship from the Japan Society for the Promotion of Science for Japan-U.S. Cooperative Research Program and to whom correspondence should be addressed. 3 Abbreviations used: Chl, chlorophyll; Rchl, bacteriochlorophyll; Chlide, chlorophyllide; DV, divinyl; MV, monovinyl; ODS, octadecyl silica; Pchl, protochlorophyll; Pchlide, protochlorophyllide.
493
routinely to study Chl biosynthesis (10-12). Recently Hanamoto and Castelfranco ( 13) have reported the separation of MV and DV Pchlide and Chlide using an ODS column and an ion-pair elution method. These HPLC analyses have been performed largely with chemically bonded silica particles as the stationary phase (3- 13). This support is superior to the separation of esterified Chl species (8), but nonesterified Chls cannot be separated without using ion-pairing (13) or ionsuppression methods (9). Polyethylene powder has been used as the support in thin-layer (14) and column chromatography (15) for the separation of plant pigments. It has also been shown to function effectively as a stationary phase in reversedphase HPLC. Chow et al. have achieved good separations of Bchls c using a preparative polyethylene column and aqueous acetone as mobile phase (16). They pointed out the advantages of polyethylene as the pack-
0003-2697187 $3.00 Copyright 0 1987 by Academic Pres, Inc. All rights of reproduction in any form reserved.
494
SHIOI AND BEALE
ing material for HPLC in terms of low cost and durability. The present report describes an HPLC system using a column packed with polyethylene powder, which enables the separation of Chls and their derivatives including MV and DV species by a simple elution with aqueous acetone. This method is simply scaled up for preparative work and allows high resolution and versatility in the preparation of plant pigments. MATERIALS
AND METHODS
Chromatography. HPLC was carried out with a Shimadzu LC-3A chromatograph using a polyethylene column. The column was encased in an acrylic plastic water jacket, and the temperature was controlled by an Ecsacal Model EX- 100 recirculating water bath. Chls were eluted with the indicated percentages of acetone in water at a flow rate of 0.2 ml/min at 20°C unless otherwise indicated. Separated pigments were detected fluorometrically and recorded as described previously (8,9). The wavelengths used for the detection of the pigments are described in the figure legends. HPLC peaks were identified by comparison of their retention times and fluorescence maxima with those obtained from authentic samples. Column packing. Polyethylene, reversedphase HPLC grade (Polysciences, Inc, Warrington, PA), was used after washing once with absolute acetone. The column (4.6 x 250 mm, 10 X 250 mm) was packed manually with dried polyethylene powder with the aid of a plastic stick. The column was then run at a pressure of 150-180 kg/cm* to compress the packing material. A void at the top of the column was packed again in a similar manner and this process was repeated several times until no void remained. The presence of a void results in a great decrease in column efficiency.
Preparation of chlorophylls. Chls a and b and Chlides a and b were prepared as described previously (8,9). A mixture of Pchlide and Pchls was extracted from 5-dayold etiolated cucumber (Cucumis sativus L.) cotyledons. MV- and DV-Chlides a were prepared from etiolated cucumber cotyledons after exposure to 1 s of white light. MV-Pchlide was partially purified from the extract of 6-day-old etiolated maize (Zea mays L.) leaves. DV-Pchlide was extracted and partially purified from the growth medium of Rhodopseudomonas sphaeroides grown in the nicotinamide-enriched medium (in preparation). Pheophytin derivatives were prepared by acid treatment of the respective pure Chls by the method of Perkins and Roberts (17). Bchl a was extracted from R. sphaeroides with acetone/methanol (712, v/v) and partially purified by DEAE-Toyopearl column chromatography (8). Chls cl and c2 were prepared and identified by polyethylene thin-layer chromatography according to the method of Jeffrey (14). Spectrophotometric measurements were performed with a Shimadzu UV-240 spectrophotometer and a Hitachi 650-60 fluorescence spectrophotometer. RESULTS
Separation Factors As with reversed-phase chromatography of other media, peak retention was greatly changed by changing the polarity of the mobile phase and the temperature. The resolution calculated from the separation of structurally similar pigments, MV- and DVPchlides, sharply increased with increasing polarity of the mobile phase and also linearly increased with decreasing column temperature (Fig. 1). This means that the resolution of the pigment separation can be controlled by selecting the mobile phase polarity and temperature. We therefore 6xed the temperature at 20°C and adjusted the polarity of the mobile phase, acetone/water mixture, to op-
CHROMATOGRAPHIC
SEPARATION
OF MONO- AND DIVINYL
I 0
1. Effects of mobile phase polarity (A) and temperature (B) on the resolution calculated from the separation of MV- and DV-Pchlide mixture. The resolution of each separation was calculated by R, = 2(fR2- fRI)/(wI + ~2). where 1~ and w are the retention times and peak widths of each pigment, respectively. (A) The pigments were eluted with the indicated percentages of acetone/ water at 2O’C. (B) The pigments were eluted with a 65% (v/v) acetone/water mixture at the indicated temperature. Flow rate was fixed at 0.2 ml/mm. Chls were detected fluorometrically using excitation and emission wavelengths at 430 and 640 nm, respectively. FIG.
timize the separation. A suitable acetone/ water concentration for the isocratic separation of Chls is presented in Table 1. Under the conditions described, Chl pigments are TABLE
1
OPTIMAL ACETONE/WATER PROPORTIONS FOR IsoCRATIC SEPARATION OF VARIOUS CHLOROPHYLLS AND THEIR DERIVATIVES
Chlorophyll” Chlorophyll u/b Pheophytin u/b Chlorophyllide a/b Pheophorbide a/b Chlorophyll c Protochlorophyll Protochlorophylhde Bacteriochlorophyll a
Acetone concentration in water (%, v/v) 15 80 50 60 67 82 65 15
LIPigments were eluted at a flow rate of 0.2 ml/min at 20°C.
I
495
CHLOROPHYLLS
I 20 Retention
I 40
I time,
I
I 60
min
FIG. 2. Elution profile of MV- and DV-Pchlide by polyethylene column HPLC. Pchls were eluted with a 65% (v/v) acetone/water mixture at a flow rate of 0.2 ml/min at 2O’C. Detection conditions as in Fig. 1. 1 = Unidentified pigments; 2 = MV-Pchlide; 3 = DVPchlide.
separated within 40 min without leading to the formation of artifacts.
Separation of Nonesterijied Chlorophylls MV- and DV-Pchlides. Figure 2 shows the separation profile of a mixture of MV- and DV-Pchlides on a packed polyethylene column using 65% acetone/water at 20°C. These pigments were completely separated in less than 38 min using a simple aqueous acetone eluant. MV- (peak 2) and DVPchlide (peak 3) were identified both by in situ fluorescence excitation and emission maxima and by comparison of the retention times with those of authentic samples. Peak 1 group was not identified, but probably consists of degradation products of the Pchlides. Chls cl and ~2. The elution profile of Chls cl and c2 is shown in Fig. 3. The two pigments were completely separated using a polyethylene column in less than 30 min with 67% acetone/water (v/v) at 20°C. Peaks 2 and 3 were identified as Chls cI and cz in terms of the retention times of each authentic sample. Peak 1 is an unidentified degradation product. MV- and DV-Chlides a and b. Figure 4 shows the separation of naturally occurring
496
SHIOI AND BEALE
1 0
I
1 20 Retention
I
I 40 time,
I
I 60
min
PIG. 3. Separation profile of Chls c by polyethylene column HPLC. The pigments were eluted with an acetone/water mixture (67’S, v/v) at a flow rate of 0.2 ml/ min at 2O’C. The pigments were detected Buorometritally using excitation and emission wavelengths at 430 and 650 nm, respectively. 1 = Unidentified pigment; 2 =Chlc,;3=Chlcz.
Chlides extracted from etiolated cucumber cotyledons after 1 s of illumination. MVChlide a (peak 4) and DV-Chlide a (peak 5) were completely separated in less than 25 min with an eluant of 50% acetone/water (v/v) at 20°C. These Chlides were identified by their in situ fluorescence excitation and emission maxima (13). Also, the retention time of MV-Chlide a ( 16.1 min) was consistent with that of MV-Chlide a prepared by the action of chlorophyllase on Chl a. Chlide b (retention time, 12.2 min) and MV-Chlide a were also clearly separated under these conditions, although the resolution of degradation products of Chlide b, probably 1O-hydroxy( 13’-hydroxy)-MV-Chlide b, is poor (data not shown). The retention time of peak 3 (12.2 min) was similar to that of authentic Chlide b. In situ fluorescence excitation and emission maxima occurred at 424 and 669 nm, respectively. These spectroscopic properties were inconsistent with Chlide b. Peak 3 was therefore not identified at present and unretained pigments, peaks 1 and 2, are also unknown. In the separation of nonesterified Chls, unidentified peaks eluting with a retention time of 7- 10 min are due to polar impurities in-
eluding degradation products (Figs. 2-4). Nonesterified Chls are generally much more subject to degradation such as oxidation than are its ester forms. At present, however, the identification of these pigments is difficult because little information is available concerning the spectral and structural properties of these minor pigments. As can be seen from the chromatographic behavior of this system (Fig. l), a more polar mobile solution is necessary to achieve better separation of the pigments. At a concentration less than 50% acetone/water, however, Chlides were increasingly retained on the support and no quantitative analysis was performed. In addition, nonpolar pigments in a mixture were strongly retained on the support under polar elution systems, which are designed for the separation of polar pigments like Chlides. In these situations, the column capacity was decreased by the adsorption of the pigments to the support. However, the column can be fully regenerated, and all adsorbed pigments recovered, by washing with 3-5 vol of absolute acetone. Separation of Esterljied Chlorophylls Chls and pheophytins. The elution profile of Chls and pheophytins esterified with phy-
I 0
I
I 20 Retention
I
I
I 40 time,
, 60
min
FIG. 4. Elution profile of Chlides extracted from etiolated cucumber cotyledons after 1 s of illumination. The pigments were eluted with a 50% acetone/water mixture (v/v) at a flow rate of 0.2 ml/min at 20°C. Chlides were detected by fluorescence measurement (excitation 430 nm; emission 660 nm). 1 to 3 = Unidentified pigments; 4 = MV-Chlide a; 5 = DV-Chlide a.
CHROMATOGRAPHIC
SEPARATION
OF MONO-
AND DIVINYL
2 3
I-AL 3
4
1
497
CHLOROPHYLLS
4
2
I
L ,i I 0
I
5
ILL I 20 Retention
I 40
I time,
I
I 60
r 1 0
min
FIG. 5. Separation of mixed Chls and pheophytins esterified with phytol by polyethylene column HPLC. The pigments were eluted with an acetone/water mixture (80%, v/v) at a tlow rate of0.2 ml/mitt at 20°C. The pigments were detected fluorometrically using excitation and emission wavelengths at 440 and 650 nm, respectively. 1 = Chl b; 2 = Chl a; 3 = pheophytin b; 4 = pheophytin a.
I
I 20 Retention
I
I 40
time,
I
I 60
min
FIG. 6. Elution profile by polyethylene column HPLC of Pchlide and Pchls extracted from etiolated cucumber cotyledons. The pigments were eluted with an acetone/ water mixture (82%, v/v) at flow rate of 0.2 ml/min at 20°C. Pigment peaks were detected by tluorescence measurement (excitation 430 nm; emission 640 nm). 1 = MV-, DV-P&ides mixture; 2 = PChlgmnyMnior; 3 = PChldihydrogennylscraniol;
4
=
PChl,trshyd~ogcnm~~i~i;
5
= Pchlph90r.
to1 is shown in Fig. 5. These pigments were completely separated with 80% acetone/ water (v/v) at 20°C. Spectral properties and retention times were consistent with their identification as Chl b (peak l), Chl a (peak 2), pheophytin b (peak 3), and pheophytin a (peak 4). The number of theoretical plates (N), a measure of column efficiency, was calculated to be 1444 for Chl a and 2036 for Chl b, respectively, under these conditions. For the separation of Chls a and b esterified with different alcohols (&lo), a more polar mobile phase, 75% acetone/water mixture, should be used (see Table 1). Pchls. Figure 6 shows the elution profile of Pchlide and Pchls extracted from etiolated cucumber cotyledons. Although Pchlide species were not retained, but eluted at the front as a mixture, four Pchls esterified with different CzO alcohols were completely separated with 82% acetone/water (v/v) at 20°C. As reported previously (11,12), the content of Pchl phytolin etiolated cucumber cotyledons was very low compared to three other Pchls. Identification of the pigments was achieved as described previously (8,ll).
Bchl a. Four Bchl a species with different CzO alcohols and bacteriopheophytin aphyl,,i were completely separated within 40 min with 75% acetone/water (v/v) (Fig. 7). Separated peaks of Bchls a have been previously
L 0
1
I 20 Retention
I
I 40 time,
I
I 60
min
FIG 7. Separation of Rchls a ester&d with different Cze alcohols and bacteriopheophytin a by polyethylene column HPLC. The pigments were eluted with an acetone/water mixture (790, v/v) at a flow rate of 0.2 ml/ min at 20°C. R&Is were detected by fluorescence measurement (excitation 370 nm; emission 780 nm). I = Bchl q-,ybrpnio~; 2 = Rchl &aYWtio~; 3 = Rchl atcvnhydroenrnytgcranial; 4 = Bchl cr$,@; 5 = bacteriopheophytin aphyfol .
498
SHIOI AND BEALE
lWI b b
20 0
50
100
FIG. 8. Calibration curves of DV-Pchlide separated by HPLC using polyethylene column. The ordinate represents the relative areas of the peaks determined by fluorescence measurements. The abscissa indicates the picomoles of DV-Pchlide used Chromatographic conditions are described in the text and in the legend to Fig. 1, except elution was with 70% acetone/water (v/v) instead of 65% acetone/water (v/v). Curve a: O-2.0 pmol range; curve b: 0- IO0 pm01 range.
identified by HPLC using ODS column and gas chromatography (see Ref. (8)). Bacteriopheophytin a was identified both by in situ excitation and emission maxima and by comparison of the retention time with an authentic sample. Quantitation of Chls. The recovery of the pigments from the column, i.e., the integrated area of peaks as measured by fluorescence emission, showed a linear relationship over a wide range of Chl concentrations (Fig. 8). The lowest detection level was determined to be 30 fmol for DV-Pchlide. Changes in the injection volume in the range of l-20 ~1 did not affect the quantitation of DV-Pchlide. DISCUSSION
The method described in this report provides rapid separation and identification of most Chls, including MV- and DV-Chlide species, in the plant extracts. This technique also allows the quantitative separation of
Chls at femtomole levels with a negligible loss. The elution system employs simple aqueous acetone, and no ion-suppressing or ion-pairing reagent is required for the separation of nonesterified Chls, as contrasted with previous methods (9,13) using chemically bonded silica material as the stationary phase. As has been pointed out by Chow et al. ( 16), the advantages of the polyethylene column are extreme inertness and a low cost of packing material, which permit the application of this technique to preparative purpose. Preparative use of this system is especially suitable for the separation of nonesterified Chls. Such separations are possible with chemically bonded silica columns only with the addition of an ion-suppressing or ionpairing reagent. We routinely used the semipreparative column (10 X 250 mm) for preparation of the standard pigments under the conditions described here. We have previously used an ODS column and the ion-suppression method for the separation of nonesterified Chls including Chl c (9). However, we were unable to achieve the separation of MV and DV species, and the resolution of Chls cl and c2 was unsatisfactory. Hanamoto and Castelfranco have achieved the separation of MV- and DVPchlide and Chlides using an ODS column with an ion-pairing method ( 13). The separation of the pigment by their method is time consuming and the elution system was complex compared to our present technique. The reproducibility of the separation and the column efficiency of this method for estermed Chls are inferior to those of the previous technique (8). The low reproducibility of the present method for esterified Chls is probably due to a sharp change of peak retention with slight differences in polarity of mobile phase (cf. Fig. 1). However, the present method shows high versatility and allows the separation of mixtures of structurally similar pigments containing both polar
CHROMATOGRAPHIC
SEPARATION
OF MONO-
and nonnolar soecies with a simole I I . elution of aqueous acetone. It is concluded that HPLC on polyethylene is a superior alternative to the known reversed-phase HPLC for the analysis and quantitation of Chls and related pigments. REFERENCES 1. Mattheis, J. R., and Rebeiz, C. A. (1977) J. Biol. Chem. 252,4022-4024. 2. Tripathy, B. C., and Rebeiz, C. A. (1985) Anal. Biothem. 149,43-6 1. 3. Schoch, S., Lempert, U., Wieschhoff, H., and Scheer, H. (1978) J. Chromatogr. 157, 357-364. 4. Braumann, T., and Grimme, L. H. (198 I) Biochim. Biophys. Acta 637, 8- 17. 5. Burke, S., and Aronoff, S. (198 1) Anal. Biochem. 114,367-370. 6. Eskins, K., and Harris, L. (198 1) Photochem. Photobiol. 33, 131-133.
AND DIVINYL
CHLOROPHYLLS
499
7. Scholz, B., and Ballschmiter, K. (1981) J Chromatogr. 208, 148-155. 8. Shioi, Y., Fukae, R., and Sass, T. (1983) Biochim. Biophys. Acta 722, 72-79. 9. Shioi, Y., Doi, M., and Sass, T. (1984) J. Chromato,er. 298. 141-149. 10. Shio; Y., and Sasa, T. (1983) B&him. Biophys. Acta 756, 127-131. 11. Shioi, Y., and Sass, T. (1983) Arch. Biochem. Biophys. 220,286-292. 12. Shioi, Y., and Sass, T. (1983) Plant Cell Physiol. 24, 835-840. 13. Hanamoto, C. M., and Castelfranco, P. A. (1983) Plant Physiol. 73, 79-8 1. 14. Jeffrey, S. W. (1972) Biochim. Biophys. Acta 279, 15-33. 15. Anderson, A. F. H., and Calvin, M. (1962) Nature (London) 194,285-286. 16. Chow, H.-C., Caple, M. B., and Strouse, C. E. (1978) J. Chromatogr. 151, 357-362. 17. Perkins, H. J., and Roberts, D. W. A. (1962) Biochim. Biophys. Acta 58,486-498.
BIOCHEMISTRY
162,493-499
(1987)
Polyethylene-Based High-Performance Liquid Chromatography Chloroplast Pigments: Resolution of Mono- and Divinyl Chlorophyllides and Other Pigment Mixtures’ Yuzo Division
of Biology,
SHIOI’
AND SAMUEL
Miyazaki Medical College, Kiyotake, Miyazaki and Medicine, Brown University, Providence,
of
I. BEALE* 889-16, Japan, and *Division Rhode Island 02912
of Biology
Received October 3 1, 1986 In addition to most chlorophylls and their derivatives, monovinyl and divinyl chlorophyll species were separated by high-performance liquid chromatography, using a polyethylene column and a simple elution with aqueous acetone. Peak retention and resolution of the pigment separation were greatly increased by increasing the polarity of the mobile phase and also by decreasing the column temperature. Polyethylene chromatography showed chlorophyll separation behavior similar to that of the octadecyl silica column, but it showed no adsorption of the pigment species containing free carboxylic acid groups, enabling the complete separation of chlorophylls and their derivatives. Polyethylene is a superior alternative stationary phase to the known reversed-phase materials for chlorophyll separation and analysis. o 1987 Academic press ITIC. KEY WORDS: chlorophyll separation; HPLC; mono- and divinyl chlorophylls; HPLC, polyethylene column.
Recently, it has become apparent that chlorophyll (Chl)3-pigment pools of etiolated and greening plants are not chemically homogeneous and that these pigment pools consist of MV and DV components (1). The detection of both MV and DV forms of Pchlide has been accomplished by fluorescence spectroscopy (2), but separation techniques for these compounds have not been fully developed. HPLC is commonly used for the separation and analysis of plant and bacterial pigments (3- 13). We have used this technique ’ Part of this work was done in Japan. 2 Recipient of a fellowship from the Japan Society for the Promotion of Science for Japan-U.S. Cooperative Research Program and to whom correspondence should be addressed. 3 Abbreviations used: Chl, chlorophyll; Rchl, bacteriochlorophyll; Chlide, chlorophyllide; DV, divinyl; MV, monovinyl; ODS, octadecyl silica; Pchl, protochlorophyll; Pchlide, protochlorophyllide.
493
routinely to study Chl biosynthesis (10-12). Recently Hanamoto and Castelfranco ( 13) have reported the separation of MV and DV Pchlide and Chlide using an ODS column and an ion-pair elution method. These HPLC analyses have been performed largely with chemically bonded silica particles as the stationary phase (3- 13). This support is superior to the separation of esterified Chl species (8), but nonesterified Chls cannot be separated without using ion-pairing (13) or ionsuppression methods (9). Polyethylene powder has been used as the support in thin-layer (14) and column chromatography (15) for the separation of plant pigments. It has also been shown to function effectively as a stationary phase in reversedphase HPLC. Chow et al. have achieved good separations of Bchls c using a preparative polyethylene column and aqueous acetone as mobile phase (16). They pointed out the advantages of polyethylene as the pack-
0003-2697187 $3.00 Copyright 0 1987 by Academic Pres, Inc. All rights of reproduction in any form reserved.
494
SHIOI AND BEALE
ing material for HPLC in terms of low cost and durability. The present report describes an HPLC system using a column packed with polyethylene powder, which enables the separation of Chls and their derivatives including MV and DV species by a simple elution with aqueous acetone. This method is simply scaled up for preparative work and allows high resolution and versatility in the preparation of plant pigments. MATERIALS
AND METHODS
Chromatography. HPLC was carried out with a Shimadzu LC-3A chromatograph using a polyethylene column. The column was encased in an acrylic plastic water jacket, and the temperature was controlled by an Ecsacal Model EX- 100 recirculating water bath. Chls were eluted with the indicated percentages of acetone in water at a flow rate of 0.2 ml/min at 20°C unless otherwise indicated. Separated pigments were detected fluorometrically and recorded as described previously (8,9). The wavelengths used for the detection of the pigments are described in the figure legends. HPLC peaks were identified by comparison of their retention times and fluorescence maxima with those obtained from authentic samples. Column packing. Polyethylene, reversedphase HPLC grade (Polysciences, Inc, Warrington, PA), was used after washing once with absolute acetone. The column (4.6 x 250 mm, 10 X 250 mm) was packed manually with dried polyethylene powder with the aid of a plastic stick. The column was then run at a pressure of 150-180 kg/cm* to compress the packing material. A void at the top of the column was packed again in a similar manner and this process was repeated several times until no void remained. The presence of a void results in a great decrease in column efficiency.
Preparation of chlorophylls. Chls a and b and Chlides a and b were prepared as described previously (8,9). A mixture of Pchlide and Pchls was extracted from 5-dayold etiolated cucumber (Cucumis sativus L.) cotyledons. MV- and DV-Chlides a were prepared from etiolated cucumber cotyledons after exposure to 1 s of white light. MV-Pchlide was partially purified from the extract of 6-day-old etiolated maize (Zea mays L.) leaves. DV-Pchlide was extracted and partially purified from the growth medium of Rhodopseudomonas sphaeroides grown in the nicotinamide-enriched medium (in preparation). Pheophytin derivatives were prepared by acid treatment of the respective pure Chls by the method of Perkins and Roberts (17). Bchl a was extracted from R. sphaeroides with acetone/methanol (712, v/v) and partially purified by DEAE-Toyopearl column chromatography (8). Chls cl and c2 were prepared and identified by polyethylene thin-layer chromatography according to the method of Jeffrey (14). Spectrophotometric measurements were performed with a Shimadzu UV-240 spectrophotometer and a Hitachi 650-60 fluorescence spectrophotometer. RESULTS
Separation Factors As with reversed-phase chromatography of other media, peak retention was greatly changed by changing the polarity of the mobile phase and the temperature. The resolution calculated from the separation of structurally similar pigments, MV- and DVPchlides, sharply increased with increasing polarity of the mobile phase and also linearly increased with decreasing column temperature (Fig. 1). This means that the resolution of the pigment separation can be controlled by selecting the mobile phase polarity and temperature. We therefore 6xed the temperature at 20°C and adjusted the polarity of the mobile phase, acetone/water mixture, to op-
CHROMATOGRAPHIC
SEPARATION
OF MONO- AND DIVINYL
I 0
1. Effects of mobile phase polarity (A) and temperature (B) on the resolution calculated from the separation of MV- and DV-Pchlide mixture. The resolution of each separation was calculated by R, = 2(fR2- fRI)/(wI + ~2). where 1~ and w are the retention times and peak widths of each pigment, respectively. (A) The pigments were eluted with the indicated percentages of acetone/ water at 2O’C. (B) The pigments were eluted with a 65% (v/v) acetone/water mixture at the indicated temperature. Flow rate was fixed at 0.2 ml/mm. Chls were detected fluorometrically using excitation and emission wavelengths at 430 and 640 nm, respectively. FIG.
timize the separation. A suitable acetone/ water concentration for the isocratic separation of Chls is presented in Table 1. Under the conditions described, Chl pigments are TABLE
1
OPTIMAL ACETONE/WATER PROPORTIONS FOR IsoCRATIC SEPARATION OF VARIOUS CHLOROPHYLLS AND THEIR DERIVATIVES
Chlorophyll” Chlorophyll u/b Pheophytin u/b Chlorophyllide a/b Pheophorbide a/b Chlorophyll c Protochlorophyll Protochlorophylhde Bacteriochlorophyll a
Acetone concentration in water (%, v/v) 15 80 50 60 67 82 65 15
LIPigments were eluted at a flow rate of 0.2 ml/min at 20°C.
I
495
CHLOROPHYLLS
I 20 Retention
I 40
I time,
I
I 60
min
FIG. 2. Elution profile of MV- and DV-Pchlide by polyethylene column HPLC. Pchls were eluted with a 65% (v/v) acetone/water mixture at a flow rate of 0.2 ml/min at 2O’C. Detection conditions as in Fig. 1. 1 = Unidentified pigments; 2 = MV-Pchlide; 3 = DVPchlide.
separated within 40 min without leading to the formation of artifacts.
Separation of Nonesterijied Chlorophylls MV- and DV-Pchlides. Figure 2 shows the separation profile of a mixture of MV- and DV-Pchlides on a packed polyethylene column using 65% acetone/water at 20°C. These pigments were completely separated in less than 38 min using a simple aqueous acetone eluant. MV- (peak 2) and DVPchlide (peak 3) were identified both by in situ fluorescence excitation and emission maxima and by comparison of the retention times with those of authentic samples. Peak 1 group was not identified, but probably consists of degradation products of the Pchlides. Chls cl and ~2. The elution profile of Chls cl and c2 is shown in Fig. 3. The two pigments were completely separated using a polyethylene column in less than 30 min with 67% acetone/water (v/v) at 20°C. Peaks 2 and 3 were identified as Chls cI and cz in terms of the retention times of each authentic sample. Peak 1 is an unidentified degradation product. MV- and DV-Chlides a and b. Figure 4 shows the separation of naturally occurring
496
SHIOI AND BEALE
1 0
I
1 20 Retention
I
I 40 time,
I
I 60
min
PIG. 3. Separation profile of Chls c by polyethylene column HPLC. The pigments were eluted with an acetone/water mixture (67’S, v/v) at a flow rate of 0.2 ml/ min at 2O’C. The pigments were detected Buorometritally using excitation and emission wavelengths at 430 and 650 nm, respectively. 1 = Unidentified pigment; 2 =Chlc,;3=Chlcz.
Chlides extracted from etiolated cucumber cotyledons after 1 s of illumination. MVChlide a (peak 4) and DV-Chlide a (peak 5) were completely separated in less than 25 min with an eluant of 50% acetone/water (v/v) at 20°C. These Chlides were identified by their in situ fluorescence excitation and emission maxima (13). Also, the retention time of MV-Chlide a ( 16.1 min) was consistent with that of MV-Chlide a prepared by the action of chlorophyllase on Chl a. Chlide b (retention time, 12.2 min) and MV-Chlide a were also clearly separated under these conditions, although the resolution of degradation products of Chlide b, probably 1O-hydroxy( 13’-hydroxy)-MV-Chlide b, is poor (data not shown). The retention time of peak 3 (12.2 min) was similar to that of authentic Chlide b. In situ fluorescence excitation and emission maxima occurred at 424 and 669 nm, respectively. These spectroscopic properties were inconsistent with Chlide b. Peak 3 was therefore not identified at present and unretained pigments, peaks 1 and 2, are also unknown. In the separation of nonesterified Chls, unidentified peaks eluting with a retention time of 7- 10 min are due to polar impurities in-
eluding degradation products (Figs. 2-4). Nonesterified Chls are generally much more subject to degradation such as oxidation than are its ester forms. At present, however, the identification of these pigments is difficult because little information is available concerning the spectral and structural properties of these minor pigments. As can be seen from the chromatographic behavior of this system (Fig. l), a more polar mobile solution is necessary to achieve better separation of the pigments. At a concentration less than 50% acetone/water, however, Chlides were increasingly retained on the support and no quantitative analysis was performed. In addition, nonpolar pigments in a mixture were strongly retained on the support under polar elution systems, which are designed for the separation of polar pigments like Chlides. In these situations, the column capacity was decreased by the adsorption of the pigments to the support. However, the column can be fully regenerated, and all adsorbed pigments recovered, by washing with 3-5 vol of absolute acetone. Separation of Esterljied Chlorophylls Chls and pheophytins. The elution profile of Chls and pheophytins esterified with phy-
I 0
I
I 20 Retention
I
I
I 40 time,
, 60
min
FIG. 4. Elution profile of Chlides extracted from etiolated cucumber cotyledons after 1 s of illumination. The pigments were eluted with a 50% acetone/water mixture (v/v) at a flow rate of 0.2 ml/min at 20°C. Chlides were detected by fluorescence measurement (excitation 430 nm; emission 660 nm). 1 to 3 = Unidentified pigments; 4 = MV-Chlide a; 5 = DV-Chlide a.
CHROMATOGRAPHIC
SEPARATION
OF MONO-
AND DIVINYL
2 3
I-AL 3
4
1
497
CHLOROPHYLLS
4
2
I
L ,i I 0
I
5
ILL I 20 Retention
I 40
I time,
I
I 60
r 1 0
min
FIG. 5. Separation of mixed Chls and pheophytins esterified with phytol by polyethylene column HPLC. The pigments were eluted with an acetone/water mixture (80%, v/v) at a tlow rate of0.2 ml/mitt at 20°C. The pigments were detected fluorometrically using excitation and emission wavelengths at 440 and 650 nm, respectively. 1 = Chl b; 2 = Chl a; 3 = pheophytin b; 4 = pheophytin a.
I
I 20 Retention
I
I 40
time,
I
I 60
min
FIG. 6. Elution profile by polyethylene column HPLC of Pchlide and Pchls extracted from etiolated cucumber cotyledons. The pigments were eluted with an acetone/ water mixture (82%, v/v) at flow rate of 0.2 ml/min at 20°C. Pigment peaks were detected by tluorescence measurement (excitation 430 nm; emission 640 nm). 1 = MV-, DV-P&ides mixture; 2 = PChlgmnyMnior; 3 = PChldihydrogennylscraniol;
4
=
PChl,trshyd~ogcnm~~i~i;
5
= Pchlph90r.
to1 is shown in Fig. 5. These pigments were completely separated with 80% acetone/ water (v/v) at 20°C. Spectral properties and retention times were consistent with their identification as Chl b (peak l), Chl a (peak 2), pheophytin b (peak 3), and pheophytin a (peak 4). The number of theoretical plates (N), a measure of column efficiency, was calculated to be 1444 for Chl a and 2036 for Chl b, respectively, under these conditions. For the separation of Chls a and b esterified with different alcohols (&lo), a more polar mobile phase, 75% acetone/water mixture, should be used (see Table 1). Pchls. Figure 6 shows the elution profile of Pchlide and Pchls extracted from etiolated cucumber cotyledons. Although Pchlide species were not retained, but eluted at the front as a mixture, four Pchls esterified with different CzO alcohols were completely separated with 82% acetone/water (v/v) at 20°C. As reported previously (11,12), the content of Pchl phytolin etiolated cucumber cotyledons was very low compared to three other Pchls. Identification of the pigments was achieved as described previously (8,ll).
Bchl a. Four Bchl a species with different CzO alcohols and bacteriopheophytin aphyl,,i were completely separated within 40 min with 75% acetone/water (v/v) (Fig. 7). Separated peaks of Bchls a have been previously
L 0
1
I 20 Retention
I
I 40 time,
I
I 60
min
FIG 7. Separation of Rchls a ester&d with different Cze alcohols and bacteriopheophytin a by polyethylene column HPLC. The pigments were eluted with an acetone/water mixture (790, v/v) at a flow rate of 0.2 ml/ min at 20°C. R&Is were detected by fluorescence measurement (excitation 370 nm; emission 780 nm). I = Bchl q-,ybrpnio~; 2 = Rchl &aYWtio~; 3 = Rchl atcvnhydroenrnytgcranial; 4 = Bchl cr$,@; 5 = bacteriopheophytin aphyfol .
498
SHIOI AND BEALE
lWI b b
20 0
50
100
FIG. 8. Calibration curves of DV-Pchlide separated by HPLC using polyethylene column. The ordinate represents the relative areas of the peaks determined by fluorescence measurements. The abscissa indicates the picomoles of DV-Pchlide used Chromatographic conditions are described in the text and in the legend to Fig. 1, except elution was with 70% acetone/water (v/v) instead of 65% acetone/water (v/v). Curve a: O-2.0 pmol range; curve b: 0- IO0 pm01 range.
identified by HPLC using ODS column and gas chromatography (see Ref. (8)). Bacteriopheophytin a was identified both by in situ excitation and emission maxima and by comparison of the retention time with an authentic sample. Quantitation of Chls. The recovery of the pigments from the column, i.e., the integrated area of peaks as measured by fluorescence emission, showed a linear relationship over a wide range of Chl concentrations (Fig. 8). The lowest detection level was determined to be 30 fmol for DV-Pchlide. Changes in the injection volume in the range of l-20 ~1 did not affect the quantitation of DV-Pchlide. DISCUSSION
The method described in this report provides rapid separation and identification of most Chls, including MV- and DV-Chlide species, in the plant extracts. This technique also allows the quantitative separation of
Chls at femtomole levels with a negligible loss. The elution system employs simple aqueous acetone, and no ion-suppressing or ion-pairing reagent is required for the separation of nonesterified Chls, as contrasted with previous methods (9,13) using chemically bonded silica material as the stationary phase. As has been pointed out by Chow et al. ( 16), the advantages of the polyethylene column are extreme inertness and a low cost of packing material, which permit the application of this technique to preparative purpose. Preparative use of this system is especially suitable for the separation of nonesterified Chls. Such separations are possible with chemically bonded silica columns only with the addition of an ion-suppressing or ionpairing reagent. We routinely used the semipreparative column (10 X 250 mm) for preparation of the standard pigments under the conditions described here. We have previously used an ODS column and the ion-suppression method for the separation of nonesterified Chls including Chl c (9). However, we were unable to achieve the separation of MV and DV species, and the resolution of Chls cl and c2 was unsatisfactory. Hanamoto and Castelfranco have achieved the separation of MV- and DVPchlide and Chlides using an ODS column with an ion-pairing method ( 13). The separation of the pigment by their method is time consuming and the elution system was complex compared to our present technique. The reproducibility of the separation and the column efficiency of this method for estermed Chls are inferior to those of the previous technique (8). The low reproducibility of the present method for esterified Chls is probably due to a sharp change of peak retention with slight differences in polarity of mobile phase (cf. Fig. 1). However, the present method shows high versatility and allows the separation of mixtures of structurally similar pigments containing both polar
CHROMATOGRAPHIC
SEPARATION
OF MONO-
and nonnolar soecies with a simole I I . elution of aqueous acetone. It is concluded that HPLC on polyethylene is a superior alternative to the known reversed-phase HPLC for the analysis and quantitation of Chls and related pigments. REFERENCES 1. Mattheis, J. R., and Rebeiz, C. A. (1977) J. Biol. Chem. 252,4022-4024. 2. Tripathy, B. C., and Rebeiz, C. A. (1985) Anal. Biothem. 149,43-6 1. 3. Schoch, S., Lempert, U., Wieschhoff, H., and Scheer, H. (1978) J. Chromatogr. 157, 357-364. 4. Braumann, T., and Grimme, L. H. (198 I) Biochim. Biophys. Acta 637, 8- 17. 5. Burke, S., and Aronoff, S. (198 1) Anal. Biochem. 114,367-370. 6. Eskins, K., and Harris, L. (198 1) Photochem. Photobiol. 33, 131-133.
AND DIVINYL
CHLOROPHYLLS
499
7. Scholz, B., and Ballschmiter, K. (1981) J Chromatogr. 208, 148-155. 8. Shioi, Y., Fukae, R., and Sass, T. (1983) Biochim. Biophys. Acta 722, 72-79. 9. Shioi, Y., Doi, M., and Sass, T. (1984) J. Chromato,er. 298. 141-149. 10. Shio; Y., and Sasa, T. (1983) B&him. Biophys. Acta 756, 127-131. 11. Shioi, Y., and Sass, T. (1983) Arch. Biochem. Biophys. 220,286-292. 12. Shioi, Y., and Sass, T. (1983) Plant Cell Physiol. 24, 835-840. 13. Hanamoto, C. M., and Castelfranco, P. A. (1983) Plant Physiol. 73, 79-8 1. 14. Jeffrey, S. W. (1972) Biochim. Biophys. Acta 279, 15-33. 15. Anderson, A. F. H., and Calvin, M. (1962) Nature (London) 194,285-286. 16. Chow, H.-C., Caple, M. B., and Strouse, C. E. (1978) J. Chromatogr. 151, 357-362. 17. Perkins, H. J., and Roberts, D. W. A. (1962) Biochim. Biophys. Acta 58,486-498.