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Fluorescence of chlorophyll a in EPA
Volun~
CWNICAL
38, nurnbt?r 2
FLUORESCENCE OF CHLOROPHYLL A.W.41.
MAU
Kcccivcd
13 November
I hlard~1976
PIIYSICS LETTERS
a IN EPA
J 975
New rcsuks .are reported from the fluore~cc~~espectra of chlurophyll o iu EPA. The new spectra 3rd obtniried fur purer SIIII~~C~than hitllcrto. Previously rq-mrtccf hot band emission is shown to bc due to impurities, and ;hc supposed dimcr ~~IIoICSCCIM!~~ to stAf--absorption.
1.
introduction
Spectroscopic properties of chlorophyll a (Chl a) been extensively studied. Most spectroscopists invest&ate systems in vitro to extract information on cncrw levels and cncrgy transfer mechanisms with the intention of correlating these with the D1lJilv system. In vitro studies are attractive because the tnaterials can be isolated and detailed studies of individual species can be made. However, the field is fraught with difficulties and conflicting claims have been made. We have re-cxarnincd the spectra with particular attention to sample purity and to avoiding spectroscopic artefacts. A particular object of the sludy was to rcexamine two aspects of the fluorescence properties which have been considered as important models for the photosynthetic system in plants. The first are the recent claims [ 1,21 on the anomalous emission of Chl a. Anomalous emission (AE) was reported on the high enerw side of the normal fluorescencc of Chl a and described as vibrationally hot fluorescence by Menzel and Polles [l] and Kawabe et aI. [2]. Their experimental conditions were the following [ I,21 : a 488 nm argon laser line provided the excitation source, Chl a was dissolved in the polar solvent EYR (diethyl ether : isopentane : ethanol in 5:5:2 mixture), and fluorescence was measured at liquid nitrogen temperatures. ‘I’ke AE if genuine suggested a significant pathway for the energy transfer in porphyrins in gcncral and have
chlorophyll systems in particular; Q more detailed investigation seemed desirable. Thus it would be important to know if thz second excited singlet state (Sz) emits in addition to the vibrationally hot fluorescence. For some porphyrins emissions from S, has been rcported 131. Also, the temperature dependence of the AE from various bands could throw light on rclaxation processes. The second involves the conditions for monomer and dirncr cquilibriuru of Chl a in polar solvents which arc the subject of some contention. The fluorescence near 730 nm has been attributed to the dimeric [2,4]. On the other hand Coedhacr IS] :md Katz et al. [6] did not find Chl a nggrogation in polar solvents. A rccent report by Kaplanova and Vacek [7] also attributed the fluorescence near 730 nm in alcoholic solutions at 77 K to dimcric Chl a cvcn at concentrations below 10d5 M. In a previous study on the properties of the acridines WChave shown that the neutral forms of acridine derivatives do not dimerize in alcoholic solutions [S] , a finding which has bczn confirmed by a Russian group [9] _
2. EiFerimental
Chl3 was extracted (~Ymdeum Vu&we)
from mutant barley leaves lacked Chl b [lo]. Prepara-
which
279
Volume 38, number 2
Cl IEhilCAL PHYSICS LE?TERS
tive methods were similar to those described by Strain 1111. The barley Icaves wea: cultured in tile tempera-
ture controlled glass house of the phytotron in CSIKO in Canberra. Spectroscopic experiments were carried out within one hour of sample preparation. Mixed EPA sotvents obtained from American Instrument Co., hlaryland U.S.A., were used without further purification.
A Gary J 7 spectrophotomcter was used for absorption expcrimcnts. Excitation sources comprised a Carson argon laser or a Varian xenon lamp dispersed by a Zeiss MM 12 double monochrornator. Fluorescence spectra -.vere measured with a ?&metel- Spcx fitted with an ITT FW130 plloton?ultipIier (f-20 msponsc). A typical absorption spectrum of Chl a in EPA at 77 K is illustrated in fig. I_ As sE~own the ratio of the intensities of the first and second absorption maxima is close to unity and the absorption rninin~urrt near 480 nm is abou: 100 times weaker than the absorption maximum near 670 nm.
I-& I. 11~ absorption spectrum of chIoraphytl 77 K, cancentration
(ii) The emissions are active only when excitation energies are be!ow the Soret system. In other words radiationless relaxation from Sz to Sr is fast and its path is different from that when 48X nm excitation is involved. To distingilish between these explanations two argon laser lines at 457.9 nm and 488.0 were employcd, Fig. 2 illustrates typical results obtained using
the Iaser excitations. When the samples were freshly prepared and extensively purified the AE was very weak. Comparing the 77 K rrl~sur~Il~el~tsthe 488 nm excitation
3. Results and discussion
In order to measure the AE in both the upper vibrational region of the first excited singlet (St ) and in the S2 (Soret) region, a xenon light source dispersed near 420 mu was used. We failed to detect any luminescence other than the normal fluorescence near 680 nm throughout the tcmperaturc range 77-238 K. An upper limit of emission intensities from the S2 region and vibrational hot band of St is lo3 times weaker thart that of the normal fluorescence which has a quantum yield of 0.2. The absence of hot band fluorescences suggested two possibilities. (i) The reported emissions at higher energy to the St electronic state were from impurities either present before or created by laser excitation. The 488 nm laser line is within the weakly absorbing region of Chl a (see fig. 1) while excitation in the Soret system (waveiength less than 460 run) reduces the importance of impurities. 280’ .
in WA nt
3
1 X 14Ts hf.
generatcd
more AE us shown
in fig. 2B than
- .-__.-_ - - ._.. - _A I
_I
._-_
WAVELENGTH (nml
Fig. 2. Fluorescence spectra of cil~ornphyl~ a in WA, 2 X IO-’ hf, mcosurod at (A> curyi: --298 K with 488 nn~ excitation, cuffs 77 K witi 957.9 nrn excitation; (l3) curve -77 K wittr 488 nm excitation, curve --77 K with 488 nm excitation after one day storage in tJle dark.
Volume 38. number 2
CHEMCAL
did 457.9 nm excitation (fig. 2A). Referring back to fig. 1 we note the onset of S2 at around 460 nm. The intensity ratio of the normal fluorescence (NF) to AE (ZNF/ZAE) was found to be greater than 500 as compared with ~25 [I ] and ~2 [2] measured car!ier under identical experimental conditions. Two hours of laser excitation ill 77 K did not alter the intensity ratios. WC conclude that the AE came mainly from impurities probably present in the sample before cxcitation. Further studies on purification procedures revealed that the carotenes and xantbophylls [ 1 l] were responsible for part of the AE measured here. If Chl a was prepared from plants which contain Chl b even argon laser lines could preferentially excite Cl11b even if it was present in minute quantitics’[ lo]. Fig. 2B also compares dre fluorescences of freshly prepared and one-day-old Chl a. Since it has been concludcc! that the impurities were the main source for the AE WCdid not pursue the temperature dependence of spectral changes.
The fluorescence profiles of Chl a have been invcstigatcd in a variety of polar solvents. The earlier as-
signment of the 680 nm band to the monomer and the 730 nm band to the dimer was based on the fact that the intensity ratios of the two bands 1730/16,0 incrcascs as the conccntrzrtion of Chi :I increases and
as the temperature decreases [2,4,7]. There appears to bc little doubt that the dimers show insignifi-
also
cant fluorescence at room tcmpcratures [4], and our experiments were made at 77 IS. For convenience we used EPA as solvent. To undertake this study we have varied (i) the path length of the fluoresccncc cells, (ii) the wavelengths of excitations and (iii) the optical geometry. Fig. 3 illustrates the fluorescence spectra. Using a particuiar concentration of Cll a and
conventional right angle excitation (curve 1) we found a considerably higher ratio of intensities (1730/f~80) than for frontal excitation (curve 2). If only the wavelengths of excitation was changed we found that in this cxc Z7so/Z6ao was lower for curve 3 than curve 2. By increasing the concentration of Chl a one hundred-fold and decreasing the cell path fifty-fold we gencratcd curve 4. There was very little difference in fluores-
cence profiles between curves 3 and 4 as shown in fig. 3.
1 hIarch 1976
PHYSICSLETTERS
WAVELENGTH (m-n) Pig. 3. Fluorcsccnce spectra of chlorophyll a in EPA at 77 K. Curve 1. right angle excitation near 480 nm, 5 X lO* hl in 10 mm cuvcttc; CUNC2, frontal excitation near 480 nm, 5 X 10% hEin ! mm ccl!; curve 3, frontal excitation near 440 nm, 5 X IO-” ht in 1 mm cell; cuwc 4, frontd excitation near 440 mn, 5 X lo4 M in 0.02 mm cell.
These observations can be rationalized in the following way. Frontal excitation (as opposed to right angle excitation), excitation within the Sorct band (rather than the weakly absorbing region), and the use of very thin cells, all minimize self-absorption of which shifts the posithe O-O band. Self-absorption, tion and.lowers the intensity of the O-O band, can cxplain the concen trztion and temperature dependence of the fluorescence profiles observed carlicr [2,4,7]. Fluoresccnccs near 730 nm cannot be assigned to dimer emission, since curve 1 obtained with a solution two orders of magnitude lower in concentration
cannot be associated with a greater concentration of dimers than curve 4.
4. Conclusion The spectrum of pure Chl a shows no evidence of hot band fluorescence, and does not support the associated energy transfer pathway. In the solution of Gil a as for most other molecules, emission is from the lowest excited state. We conclude aiso that in solutions up to 10-S M of Chl a in EPA, and in the temperature range 77-298 K, fluorescence from
timers is negligible. Acknowledgement I wish to thank Dr. David Coodchild for the barley
Volume 38; number 2
leaves and
Mt.M.Pusa for
the preparation of pure
&a.
[I j E.R. hfcnzel and J-S. Pofies. Chum. Phvs. Lcttcrs
24
545.
K. Kawrrbe, M. Nacmura and Y. Matsukawa. Tccbnol. Rcpt. Osaka Univ. 20 (1970) 665. hf. Gontcmman, in: Excited states of matter, cd. C.W. Shoppec, (Cmd. Studies Tex3r. Tech. Univ., 1973) p. 63. S.B. Broyde and S.S. Krody, J. Chem. Phys. 46 (1967) 3334, and rcfcrcnccs therein. J.C. Cotdhccr, in: The chioropItylls, cds. L.P. Vernon and G.K. Sccly (Academic I’re
282
[6J 1-J. Katz, K-C. Dougherty and L.J. Boucher, in: The chlo~opl;yIls, cds. L.P. Vernon and G.R. Seely (Academic Press, New York, 1966). [7] hi. Kaplanova and K. Vacek, Photochcm. Photobiol. 20 ci974
References (1974)
.
1 Mar& 1976
CHEMICAL PHYSICS LE”iTERS
371.
_181. J. Wrguson and A.W.H. hfau, Chcm. Phys. Letters 17 (1972) 543. L-H. Rykovskaya. K-i. Pcrsonov and B.M. Khatfamov. Chem. Piiys. Lcttcrs 27 (1974) 80. N.K. ~o~~ni~n and S.W.Thome, Biocbim. Biopbys. Acta 253 (1971) 222; N.K. Rnardman anti H.R. Hi&kin, Biochim. Mophys.
Acta :26 (1966) 189. H.H. Strain, Chloroplast
Pigments and Cluomatographic Analysis, f’en~isyiv~nj3 State Univ., U~liv~rsity Park, Pa. (1958).
CWNICAL
38, nurnbt?r 2
FLUORESCENCE OF CHLOROPHYLL A.W.41.
MAU
Kcccivcd
13 November
I hlard~1976
PIIYSICS LETTERS
a IN EPA
J 975
New rcsuks .are reported from the fluore~cc~~espectra of chlurophyll o iu EPA. The new spectra 3rd obtniried fur purer SIIII~~C~than hitllcrto. Previously rq-mrtccf hot band emission is shown to bc due to impurities, and ;hc supposed dimcr ~~IIoICSCCIM!~~ to stAf--absorption.
1.
introduction
Spectroscopic properties of chlorophyll a (Chl a) been extensively studied. Most spectroscopists invest&ate systems in vitro to extract information on cncrw levels and cncrgy transfer mechanisms with the intention of correlating these with the D1lJilv system. In vitro studies are attractive because the tnaterials can be isolated and detailed studies of individual species can be made. However, the field is fraught with difficulties and conflicting claims have been made. We have re-cxarnincd the spectra with particular attention to sample purity and to avoiding spectroscopic artefacts. A particular object of the sludy was to rcexamine two aspects of the fluorescence properties which have been considered as important models for the photosynthetic system in plants. The first are the recent claims [ 1,21 on the anomalous emission of Chl a. Anomalous emission (AE) was reported on the high enerw side of the normal fluorescencc of Chl a and described as vibrationally hot fluorescence by Menzel and Polles [l] and Kawabe et aI. [2]. Their experimental conditions were the following [ I,21 : a 488 nm argon laser line provided the excitation source, Chl a was dissolved in the polar solvent EYR (diethyl ether : isopentane : ethanol in 5:5:2 mixture), and fluorescence was measured at liquid nitrogen temperatures. ‘I’ke AE if genuine suggested a significant pathway for the energy transfer in porphyrins in gcncral and have
chlorophyll systems in particular; Q more detailed investigation seemed desirable. Thus it would be important to know if thz second excited singlet state (Sz) emits in addition to the vibrationally hot fluorescence. For some porphyrins emissions from S, has been rcported 131. Also, the temperature dependence of the AE from various bands could throw light on rclaxation processes. The second involves the conditions for monomer and dirncr cquilibriuru of Chl a in polar solvents which arc the subject of some contention. The fluorescence near 730 nm has been attributed to the dimeric [2,4]. On the other hand Coedhacr IS] :md Katz et al. [6] did not find Chl a nggrogation in polar solvents. A rccent report by Kaplanova and Vacek [7] also attributed the fluorescence near 730 nm in alcoholic solutions at 77 K to dimcric Chl a cvcn at concentrations below 10d5 M. In a previous study on the properties of the acridines WChave shown that the neutral forms of acridine derivatives do not dimerize in alcoholic solutions [S] , a finding which has bczn confirmed by a Russian group [9] _
2. EiFerimental
Chl3 was extracted (~Ymdeum Vu&we)
from mutant barley leaves lacked Chl b [lo]. Prepara-
which
279
Volume 38, number 2
Cl IEhilCAL PHYSICS LE?TERS
tive methods were similar to those described by Strain 1111. The barley Icaves wea: cultured in tile tempera-
ture controlled glass house of the phytotron in CSIKO in Canberra. Spectroscopic experiments were carried out within one hour of sample preparation. Mixed EPA sotvents obtained from American Instrument Co., hlaryland U.S.A., were used without further purification.
A Gary J 7 spectrophotomcter was used for absorption expcrimcnts. Excitation sources comprised a Carson argon laser or a Varian xenon lamp dispersed by a Zeiss MM 12 double monochrornator. Fluorescence spectra -.vere measured with a ?&metel- Spcx fitted with an ITT FW130 plloton?ultipIier (f-20 msponsc). A typical absorption spectrum of Chl a in EPA at 77 K is illustrated in fig. I_ As sE~own the ratio of the intensities of the first and second absorption maxima is close to unity and the absorption rninin~urrt near 480 nm is abou: 100 times weaker than the absorption maximum near 670 nm.
I-& I. 11~ absorption spectrum of chIoraphytl 77 K, cancentration
(ii) The emissions are active only when excitation energies are be!ow the Soret system. In other words radiationless relaxation from Sz to Sr is fast and its path is different from that when 48X nm excitation is involved. To distingilish between these explanations two argon laser lines at 457.9 nm and 488.0 were employcd, Fig. 2 illustrates typical results obtained using
the Iaser excitations. When the samples were freshly prepared and extensively purified the AE was very weak. Comparing the 77 K rrl~sur~Il~el~tsthe 488 nm excitation
3. Results and discussion
In order to measure the AE in both the upper vibrational region of the first excited singlet (St ) and in the S2 (Soret) region, a xenon light source dispersed near 420 mu was used. We failed to detect any luminescence other than the normal fluorescence near 680 nm throughout the tcmperaturc range 77-238 K. An upper limit of emission intensities from the S2 region and vibrational hot band of St is lo3 times weaker thart that of the normal fluorescence which has a quantum yield of 0.2. The absence of hot band fluorescences suggested two possibilities. (i) The reported emissions at higher energy to the St electronic state were from impurities either present before or created by laser excitation. The 488 nm laser line is within the weakly absorbing region of Chl a (see fig. 1) while excitation in the Soret system (waveiength less than 460 run) reduces the importance of impurities. 280’ .
in WA nt
3
1 X 14Ts hf.
generatcd
more AE us shown
in fig. 2B than
- .-__.-_ - - ._.. - _A I
_I
._-_
WAVELENGTH (nml
Fig. 2. Fluorescence spectra of cil~ornphyl~ a in WA, 2 X IO-’ hf, mcosurod at (A> curyi: --298 K with 488 nn~ excitation, cuffs 77 K witi 957.9 nrn excitation; (l3) curve -77 K wittr 488 nm excitation, curve --77 K with 488 nm excitation after one day storage in tJle dark.
Volume 38. number 2
CHEMCAL
did 457.9 nm excitation (fig. 2A). Referring back to fig. 1 we note the onset of S2 at around 460 nm. The intensity ratio of the normal fluorescence (NF) to AE (ZNF/ZAE) was found to be greater than 500 as compared with ~25 [I ] and ~2 [2] measured car!ier under identical experimental conditions. Two hours of laser excitation ill 77 K did not alter the intensity ratios. WC conclude that the AE came mainly from impurities probably present in the sample before cxcitation. Further studies on purification procedures revealed that the carotenes and xantbophylls [ 1 l] were responsible for part of the AE measured here. If Chl a was prepared from plants which contain Chl b even argon laser lines could preferentially excite Cl11b even if it was present in minute quantitics’[ lo]. Fig. 2B also compares dre fluorescences of freshly prepared and one-day-old Chl a. Since it has been concludcc! that the impurities were the main source for the AE WCdid not pursue the temperature dependence of spectral changes.
The fluorescence profiles of Chl a have been invcstigatcd in a variety of polar solvents. The earlier as-
signment of the 680 nm band to the monomer and the 730 nm band to the dimer was based on the fact that the intensity ratios of the two bands 1730/16,0 incrcascs as the conccntrzrtion of Chi :I increases and
as the temperature decreases [2,4,7]. There appears to bc little doubt that the dimers show insignifi-
also
cant fluorescence at room tcmpcratures [4], and our experiments were made at 77 IS. For convenience we used EPA as solvent. To undertake this study we have varied (i) the path length of the fluoresccncc cells, (ii) the wavelengths of excitations and (iii) the optical geometry. Fig. 3 illustrates the fluorescence spectra. Using a particuiar concentration of Cll a and
conventional right angle excitation (curve 1) we found a considerably higher ratio of intensities (1730/f~80) than for frontal excitation (curve 2). If only the wavelengths of excitation was changed we found that in this cxc Z7so/Z6ao was lower for curve 3 than curve 2. By increasing the concentration of Chl a one hundred-fold and decreasing the cell path fifty-fold we gencratcd curve 4. There was very little difference in fluores-
cence profiles between curves 3 and 4 as shown in fig. 3.
1 hIarch 1976
PHYSICSLETTERS
WAVELENGTH (m-n) Pig. 3. Fluorcsccnce spectra of chlorophyll a in EPA at 77 K. Curve 1. right angle excitation near 480 nm, 5 X lO* hl in 10 mm cuvcttc; CUNC2, frontal excitation near 480 nm, 5 X 10% hEin ! mm ccl!; curve 3, frontal excitation near 440 nm, 5 X IO-” ht in 1 mm cell; cuwc 4, frontd excitation near 440 mn, 5 X lo4 M in 0.02 mm cell.
These observations can be rationalized in the following way. Frontal excitation (as opposed to right angle excitation), excitation within the Sorct band (rather than the weakly absorbing region), and the use of very thin cells, all minimize self-absorption of which shifts the posithe O-O band. Self-absorption, tion and.lowers the intensity of the O-O band, can cxplain the concen trztion and temperature dependence of the fluorescence profiles observed carlicr [2,4,7]. Fluoresccnccs near 730 nm cannot be assigned to dimer emission, since curve 1 obtained with a solution two orders of magnitude lower in concentration
cannot be associated with a greater concentration of dimers than curve 4.
4. Conclusion The spectrum of pure Chl a shows no evidence of hot band fluorescence, and does not support the associated energy transfer pathway. In the solution of Gil a as for most other molecules, emission is from the lowest excited state. We conclude aiso that in solutions up to 10-S M of Chl a in EPA, and in the temperature range 77-298 K, fluorescence from
timers is negligible. Acknowledgement I wish to thank Dr. David Coodchild for the barley
Volume 38; number 2
leaves and
Mt.M.Pusa for
the preparation of pure
&a.
[I j E.R. hfcnzel and J-S. Pofies. Chum. Phvs. Lcttcrs
24
545.
K. Kawrrbe, M. Nacmura and Y. Matsukawa. Tccbnol. Rcpt. Osaka Univ. 20 (1970) 665. hf. Gontcmman, in: Excited states of matter, cd. C.W. Shoppec, (Cmd. Studies Tex3r. Tech. Univ., 1973) p. 63. S.B. Broyde and S.S. Krody, J. Chem. Phys. 46 (1967) 3334, and rcfcrcnccs therein. J.C. Cotdhccr, in: The chioropItylls, cds. L.P. Vernon and G.K. Sccly (Academic I’re
282
[6J 1-J. Katz, K-C. Dougherty and L.J. Boucher, in: The chlo~opl;yIls, cds. L.P. Vernon and G.R. Seely (Academic Press, New York, 1966). [7] hi. Kaplanova and K. Vacek, Photochcm. Photobiol. 20 ci974
References (1974)
.
1 Mar& 1976
CHEMICAL PHYSICS LE”iTERS
371.
_181. J. Wrguson and A.W.H. hfau, Chcm. Phys. Letters 17 (1972) 543. L-H. Rykovskaya. K-i. Pcrsonov and B.M. Khatfamov. Chem. Piiys. Lcttcrs 27 (1974) 80. N.K. ~o~~ni~n and S.W.Thome, Biocbim. Biopbys. Acta 253 (1971) 222; N.K. Rnardman anti H.R. Hi&kin, Biochim. Mophys.
Acta :26 (1966) 189. H.H. Strain, Chloroplast
Pigments and Cluomatographic Analysis, f’en~isyiv~nj3 State Univ., U~liv~rsity Park, Pa. (1958).