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S1 absorption of chlorophyll-a in the red region
CHEhllCAL PHYSICS LETTERS
Volume 100. number 4
S1 ABSORFTiON
16 September
1983
OF CHLOROPHYLL-a IN THE RED REGION
D. LEUPOLD Central Institute of Optics and Spectroscopy. Academy of Sciences of GDR. Rudower Chaussee. DDR-1199 Berlin. German Democratic Republic
J. EHLERT, S. OBERLANDER Institute of Mathematics. Academy of Sciences of CDR. Molwenstrasse. DDR-I 040 Berlin. German Democratic Republic
and B. WIESNER Sectionof Biolofl, Hmnboldt Unisersity of Berlin. Reinlmdtstrasse. DDR-1040 Berlin. German Democratic Republic Received 11 April 1983; in final form 24 June 1983
To clear up controversial results of excited-singlet (St) absorption of monomeric chlorophyll-a solutions in the red spectral range, non-linear absorption measurements were performed at h = 670 nm and evaluated in andytic and mmterical terms. A large St absorption cross section has been confirmed at 670 nm (OS, (670) = 0.71 ~~~(670) and excited-sinslet absorption underlined as the cduse of the anomalous short-wave emission of the Chl-a laser. The St absorption cross sections for Chl-a/dioxan in the range 800-870 nm are given.
1. Introduction Due to the central role of chlorophyll-a (Chl-a) in the process of photosynthesis. the properties of its excited electronic states are of particular interest and numerous laser-spectroscopic investigations of nonlinear effects of Chl-a have been published_ Nevertheless many of the properties of excited singlet states are unknown or controversial - this is true also for St absorption in the red spectral range (see table 1). At first a noticeable St absorption around 670 nm from the extrinsic loss-dependent data of the Chl-a laser was calculated [3] _Two years later Baugher et al_ obtained a contradictory result (“no sign of any excited singlet absorption above 500 nm” [4]). Another two years later an S, absorption spectrum was published which again showed a large absorption cross section comparable to ground-state absorption around 670 nm [5] _ Causes of these discrepancies are not given in refs. [4,5] ; they have to be looked for in
0 009-2614/83/0000-0000/s
03.00 0 1983 North-Holland
the excitation conditions (see table 1) and/or in the underlying assumptions. It may not be advantageous for investigating S, properties to use short-wave escitation pulses (i-e_ to populate S1 indirectly via higher singlets instead of directly from SO), since, particularly for Chl-a, there are indications of a wavelength dependence of fluorescent quantum yield with pulse excitation *_ We report the determination of the S, absorption cross section of Chl-a dissolved in dioxan at 670 nm with direct St population. A physical-mathematical method package for non-linear absorption was used [S--IO]_ The existence of a large S1 absorption cross section US, (670 nm) follows without any presuppositions from the curve of non-linear absorption. * At excitation with nanosecond pulses of identical power density a 25 times smaller fluorescent quantum yield was observed for X,,, = 455 nm than for &.c = 655 nm ] 161; see also a note in ref. 171.
345
Tat& I Investigations
of excited singlet absorptions
Authors
Excitation
16 September
CHEWCAL PHYSICS LETTERS
Volume 100. number 4
pulse
1983
of Chl-a in the red spectral regon Nethod
Solvent
Result
h (nm)
f!g hm
Huppert c: si. 1976 [l]
630
7 px
pump and probe pulse
ethanol
“bleaching at 660 nm”
.Arsenault et 31. 1976 [2]
694
(Q-switched)
Leupold et al. 1977 [3]
ether diosane
oS,(694 nm)= 1.26 X lo-l6 cm2 limits of St absorption cross sections
337
2.5 ns
non-linear absorption extrinsic loss variations at the CM-a-laser
337
Y.9 ns
pump and probe pulse
pvridine
355
7 PS
pump
pyridine
--
Baupiter et 31. 1979 141 Sltcpanski et al. 1981 IS]
and probe
The package of methods used represents a combination of non-linear laser spectroscopy and an estensive analytic and numerical treatment of the differential equation systems (photon transport equation and rate equations) describing the experiment. Based on esperimental findings. it allows the development of models (excitation states and their population and depopulation channels)_ the determination of unknown escitedstate parameters (absorption cross sections and relasation constants) and the calculation of path- and timedependent population density in various escited states as a function of escitation intensity. This is demonstrated below for monomeric Chl-a. In particular, the population densityjescitation energy density diagram is the basis for a quantitative test beam method (pumpand-probe-beam technique), see appendix. In a later paprr we will report the quantitative S, absorption spectrum of Chl-a/dioxan in the visible range deter-
mined this way [6] _
pulse
Chl-a was obtained from the alga Atzacysris niddans Ill]_ The non-linear absorption (intensity-dependent rransmission) of a 4 X 1 O-3 molar solution of Chl-a in diosan was investigated by dye-laser pulses variable in intensity at X = 670 nm (4x z 0.3 nm, fwbm = 2 ns). lntensity variation over 7 orders of magnitude up to a maGmum 1 OS W cm-2 was performed using calibrdted neutral filters before the sample. An analogous
336
cm2
band
set of filters after the sample made a near-null mode operation possible_ To measure the time-integrated longitudinal transmission, a laser spectroscopy detector system (Molectron) was used. Fluorescence was eliminated by cut-off filters and a large distance between sample and receiver. Fig. 1 shows the measuring result: a bleaching curve
with a plateau. Every measuring point is the mean of 10 single measurements, fig_ 1 gives a representative error width for 95% level of confidence. Checks during and after measurement did not show any measurable irreversible change of small-signal transmission_ By means of non-linear double-resonance investigations [ 121 using polarized radiation, isotropic distributions in the excited state were detected under the given measuring conditions.
3. Analytic curves
2. Experimental
between 666 and 700 nm, e.g. 0.2 < ~~~(670 nm) C 1.2 X lo-l6 “no sign of any singlet absorption above 500 nm” “a broad excited singlet absorption from 570 to 710 nm”
statements with plateau
on non-linear
transmission
If a plateau of an intensity-dependent transmission curve is defined as an area where there exist three inflection points but no extrema (this includes the interesting case that these three inflection points are far apart and transmission is nearly constant in a large environment of the central inflection point), analytic investigations show that the presence of two absorptions is necessary for the existence of a plateau and is also sufficient for suitably chosen model parameters [9]. This is true both for the case that the second ab-
Volume 100, number 4
16 September 1983
CHEMCAL PHYSICS LEIT-ERS
* .
. ...') IO7
. , .
. . ... 10'
I [w.criq Fig. 1_ X: Transmission of chlorophyll-a/dioxan solution (To = 21~5%) as a function of incidenr laser pulse (670 nmf2 ns) intensiry. o: hiafhematicai simulation of experimental curve using a model as in fig. 2c (see Jection 4).
sorption proceeds from the state the first ended in fig_ 2a and for the case that it proceeds from an energetically deeper state populated by relaxation (fig. 2b). This holds both for longitudinal and for transverse measurement. Besides it has been shown for transverse measurement in the stationary case that, by suitably chosen parameters, a plateau of any length and height can be produced and described by simple approximations. Here, the length of the plateau can be adjusted by the choice of relaxation constants, its height by the choice
of cross sections. in the non-stationary case the plateau will be shifted, in parallel to the later onset of bleaching, towards higher intensities [9]_ Last but not least analytic investigations indicate that the model necessary to describe the structure of a non-linear transmission curve is at least as large at longitudinal measurement asat transverse measurement_ (In ref. [Q] it was shown that in the stationary case, a non-linear transmission curve with longitudinal measurement can have at best as many extrema as with transverse measurement.)
4. Choice of model (energy level scheme) and numerical treatment According to section 3 the non-finear transmission of Chl-a at 670 nm is caused by a stepwise twophoton absorption according to the mode!s in fig. ?a or fig. 2b (minimum size of models). In case 3a So and S, absorptions are concerned, in case 2b So and TL absorption. (As the measuring wavelength of 670 nm lies near the maximum of So--S, absorption, on cunre
Fig. 2. Energy-level schemes for interpreting non-linear absorption of Chs-afdioxan at h = 670 nm.
347
Volume 100. rwmber 4
CHEhlIC.~l
16 September
PHYSICS LE’ITERS
its long-wave side. for singlet-singlet absorption a model of type 2b with the cycle So + S,, + S,, ++ S, can be omitted. i.e. allowance for a vibrational relaxation in St.) in case ?a. two of the four parametersare unknown: the absorption cross section US, (670) of special intetest here .md the relaxation constant kSsS, _ In case 2b. apart from the parameter ~T_~T~. all the remaining four parmnerers are known [ 13- 15]_
To determine the model effective in the given experiment and to determine the unknown excited-state parameters. the program package “Parameter identification in the spectroscopy of excited states” (PEA) 1 IO] was used *_ 1Iere. the intensity dependence of (longitudinal integral) transmission is calculated for the respective model under the given esperimental conditions - in
the first step using all known parameters and arbitrarily (in physical terms reasonably) given initial parameters for the unknown ones. Using this calculated transmission curve. an objective function valuefis determined as a measure of deviation of calculated and measured curve and this function f is then minimized in the next program step. The course of minimization shows whether the given model is sufficient and, in the confirmatory case, givesvaluesand/or limits for the unknown parameters. In the negative case (j’
does not become sufficiently small) the model has to be corrected.
Such a negative case is represented by the model 2b (So and Tt absorption) for a transmission curve according to fig. I_ (This case is particularly simple. since only one parameter is unknown_) The esperimentally found transmission course of a bleaching curve with plateau is thus caused by a two-step singlet absorption (model ?a). To determine the two
Photo”s[C&e~ lO’2 I I
Fig. 3. Standardized
population
, , ,
50” ,
.
lo” I 1 , , , ,,
1983
10=
ld6
3 1p
27
I
densities as a function of incident laser pulse (670 nm!2 ns) intensity for 4 X 10m3 molar Chl-a/ density. X: So. o: S1, 0: Tr; population
diolan solution at the cuvette end (d = 0.1 cm). A: Sa, +: S1, 0: T1; integral population density in pulse nmimum.
Volume 100. number 4
16 September 1983
CHEMICAL PHYSICS LETTERS
unknown parameters us1 (670) and ks,s,,PiSA with initial values o&O)= us and k&@s = 1011s-1 wasat first applied to model ?a and aG:ick convergence of f was reached. Then the values obtained, o&)(670) = 0.73 ~~~(670) and k!&, = 8.0X 101os-l, served as initial values in an extended model 2c, which allows for the triplet system. As expected and in accordance with the results of model 2b, there were only slight changes of parameter values in the repeated minimization process. The final parameters are 0:~(670) = 0.7 1 US (670) = 1.O X lo-l6 cm2 and kbpT = 7.6 X 101gs-r_ The respective calculated v&2 of non-linear transmission have been entered into fig. 1 for comparison with the experiment. Fig. 3 shows the intensity dependence of the main occupation densities (peak values and integrals) at the sample exit.
5. Discussion The result of a high S, absorption of Chl-a at 670 nm obtained here confirms earlier results of our group [3] and of Shepanski and Anderson [5]. The method used here avoi’ds the (one-photon) excitation of higher levels by direct S, population and thus rules out misinterpretations due to unknown deactivation paths. By applying analytic results and a numerical program package, the intensity-dependent transmission curve measured can be identified as belonging to a two-step singlet absorber and the absorption cross section uSI (670) can be determined. The present result confirms the earlier explanation of the anomalously short-wave stimulated emission of Chl-a as a consequence of S, absorption [3] and dispels doubts expressed in ref. [4]. Evidently the main cause of the discrepancy of the results of Baugher et al. [4] lies in their neglect of excited-state absorption at the pump wavelength of 337 nm. This is underlined by our experimental finding, according to which the non-linear absorption of a Chl-a solution at 337 nm shows a clear increase from 2 X 1O-5 W s cm-2 So for the evaluation of the DOD spectra excited with 2 X 1O-2 W s cm-2 [4] both knowledge of usI (337) and UT1 (337) and of the relaxation paths and constants of the states highly excited via S, +337 _ Sx , and/or T1+337 nr.r,T,, are necessary_
The high S, absorption of monomeric Chl-a in the red spectral range is an important basis for understanding the non-linear absorption and emission of chlorophyll in vivo.
Acknowledgement The authors thank Dr. P. Kis at the University of Vienna for the preparation of chlorophyll-a. The measurements described in the appendi__ were stimulated by Dr. P. Gtiber (Max-Vohner
Institute of the TU
Berlin).
Appendix.
SL absorption
in the BOB-870 range
On the basis of the abovementioned populationdensity analysis the S, absorption cross section for Chl-a/dioxan in the range 800-S70 nm was determined by an excite- and probe-beam technique. This range is of interest for instance in the investigation of the short-term kinetics of the primary redox reaction of chlorophyll in vivo. A coaxial arrangement was used for the two beams. For the excitation pulse the above dye laser was used at X = 670 run with 1, = 1.2 X 106 1%’cm-2 (at the sample input); the fluorescence of DTTC/DMSO (d= 0.2 cm; T(670 nm) = 50%) excited by the 670 nm pulse served as measuring beam. The measurement was made by monochromator (SPhl-2, Carl Zeiss Jena) by means of a multiplier and laser spectroscopy detectorsystem(Molectron). Betweenmeasuringcuvette (d = lo-’ cm; Chl-a/dioxan 4 X 10m3 mol a-l) and the entrance slit of the monochromator there was a Table 2 Sr absorption cross sections of Cl+aldiolan h (mu)
Q X 10 l6 (cm*)
800 810 820 830 840 850 860 870
0.22 0.40 0.75 1.32 0.80 0.77 1.72 1.68
349
Volume 100. number 4
CHEUICAL PHYSICS LETTERS
cut-off filter (RG 9, Schott Jena) to eliminate the 670 nxn radiation. The e~periI?lent~ll~ deterndned values of Iongitudinal integral tratwnission were corrected for the transnlission of a diosan-filled reference cuvette and the S, sbsorption cross sections calculated using the program package PEA. variant PD-2 (es&e- and probe-beam, two photon transport equations) [ 10). These are listed in table 2.
References ffuppert. P.M. Rcntzcpis and G. To&n, Biochim. Biophys. Acta 440 11976) 356. R. Arsznautt and h1.M. Denariez-Roberge. Chem. Phys. Let:ers 40 (1976) S4. D. Leupold, S Mary. P. Hoffmann, B. Hi&c and R. Ktinig,
D.
(1977) 567, J. Baughhrr. J.C. Hindman and J.J. Katz. Cbcm. RI>S
Chcll1. Phys. Letters 4s
Letters 63 (1979) 159. 3-F. Shrpanskz and R.W. Anderson Jr., Chrm. Ph!s. Letters 78 (1981) 165.
16 September 1983
161 S_ Mary, D. Leopold and J. Ehlert. to be published_ 173 M. Asano and 3-A. Ikmingstein, Chem. Phys. 57 (I 981) 1. 181 D. Leupold, 3. Ehlert and S. OberlZnder, Proceedings I. School ORLaser Pulse Fhtorometer LIF 200 (Berlin. 1982). 191 S. Oberl%nder, Analytical treatment of problems of nonlinear opkaal spectroscopy, Parts I, II. Preprint P-Math24/82 and P-Afath-O3/83 (Berlin. 1982/1983), available on request. IlO J. Ehfert and R. fahnke, PLSA pro-mm description, Parameter identification in Spectroscopy of Escited States, available on request. IllI P. Kis, Anal. Biochem. 96 (1979) 126. 1121 B. Wiesner, 8. Voigt, D. Leupold and P. Hoffmann, Proceedings IV. International Seminar on Energy Transfer in Condensed Matter (Pragttc. 1981) p. 157. rt31 H. Linschitz and I;. Sarkanen J. Am. Chem. Sot. 80 (1958) 4826. I141 G. Weber and F.W.L. Tale, J. Chem Phys. 20 (1952) 1315.
1151 A.W.H. blau and hl. Puzrt, Photocbem. Photobiol. 25 (I 977) 601.
It61 R. Jafmke and D. Leupold, Studin Biophys. 86 (1981) El.
Volume 100. number 4
S1 ABSORFTiON
16 September
1983
OF CHLOROPHYLL-a IN THE RED REGION
D. LEUPOLD Central Institute of Optics and Spectroscopy. Academy of Sciences of GDR. Rudower Chaussee. DDR-1199 Berlin. German Democratic Republic
J. EHLERT, S. OBERLANDER Institute of Mathematics. Academy of Sciences of CDR. Molwenstrasse. DDR-I 040 Berlin. German Democratic Republic
and B. WIESNER Sectionof Biolofl, Hmnboldt Unisersity of Berlin. Reinlmdtstrasse. DDR-1040 Berlin. German Democratic Republic Received 11 April 1983; in final form 24 June 1983
To clear up controversial results of excited-singlet (St) absorption of monomeric chlorophyll-a solutions in the red spectral range, non-linear absorption measurements were performed at h = 670 nm and evaluated in andytic and mmterical terms. A large St absorption cross section has been confirmed at 670 nm (OS, (670) = 0.71 ~~~(670) and excited-sinslet absorption underlined as the cduse of the anomalous short-wave emission of the Chl-a laser. The St absorption cross sections for Chl-a/dioxan in the range 800-870 nm are given.
1. Introduction Due to the central role of chlorophyll-a (Chl-a) in the process of photosynthesis. the properties of its excited electronic states are of particular interest and numerous laser-spectroscopic investigations of nonlinear effects of Chl-a have been published_ Nevertheless many of the properties of excited singlet states are unknown or controversial - this is true also for St absorption in the red spectral range (see table 1). At first a noticeable St absorption around 670 nm from the extrinsic loss-dependent data of the Chl-a laser was calculated [3] _Two years later Baugher et al_ obtained a contradictory result (“no sign of any excited singlet absorption above 500 nm” [4]). Another two years later an S, absorption spectrum was published which again showed a large absorption cross section comparable to ground-state absorption around 670 nm [5] _ Causes of these discrepancies are not given in refs. [4,5] ; they have to be looked for in
0 009-2614/83/0000-0000/s
03.00 0 1983 North-Holland
the excitation conditions (see table 1) and/or in the underlying assumptions. It may not be advantageous for investigating S, properties to use short-wave escitation pulses (i-e_ to populate S1 indirectly via higher singlets instead of directly from SO), since, particularly for Chl-a, there are indications of a wavelength dependence of fluorescent quantum yield with pulse excitation *_ We report the determination of the S, absorption cross section of Chl-a dissolved in dioxan at 670 nm with direct St population. A physical-mathematical method package for non-linear absorption was used [S--IO]_ The existence of a large S1 absorption cross section US, (670 nm) follows without any presuppositions from the curve of non-linear absorption. * At excitation with nanosecond pulses of identical power density a 25 times smaller fluorescent quantum yield was observed for X,,, = 455 nm than for &.c = 655 nm ] 161; see also a note in ref. 171.
345
Tat& I Investigations
of excited singlet absorptions
Authors
Excitation
16 September
CHEWCAL PHYSICS LETTERS
Volume 100. number 4
pulse
1983
of Chl-a in the red spectral regon Nethod
Solvent
Result
h (nm)
f!g hm
Huppert c: si. 1976 [l]
630
7 px
pump and probe pulse
ethanol
“bleaching at 660 nm”
.Arsenault et 31. 1976 [2]
694
(Q-switched)
Leupold et al. 1977 [3]
ether diosane
oS,(694 nm)= 1.26 X lo-l6 cm2 limits of St absorption cross sections
337
2.5 ns
non-linear absorption extrinsic loss variations at the CM-a-laser
337
Y.9 ns
pump and probe pulse
pvridine
355
7 PS
pump
pyridine
--
Baupiter et 31. 1979 141 Sltcpanski et al. 1981 IS]
and probe
The package of methods used represents a combination of non-linear laser spectroscopy and an estensive analytic and numerical treatment of the differential equation systems (photon transport equation and rate equations) describing the experiment. Based on esperimental findings. it allows the development of models (excitation states and their population and depopulation channels)_ the determination of unknown escitedstate parameters (absorption cross sections and relasation constants) and the calculation of path- and timedependent population density in various escited states as a function of escitation intensity. This is demonstrated below for monomeric Chl-a. In particular, the population densityjescitation energy density diagram is the basis for a quantitative test beam method (pumpand-probe-beam technique), see appendix. In a later paprr we will report the quantitative S, absorption spectrum of Chl-a/dioxan in the visible range deter-
mined this way [6] _
pulse
Chl-a was obtained from the alga Atzacysris niddans Ill]_ The non-linear absorption (intensity-dependent rransmission) of a 4 X 1 O-3 molar solution of Chl-a in diosan was investigated by dye-laser pulses variable in intensity at X = 670 nm (4x z 0.3 nm, fwbm = 2 ns). lntensity variation over 7 orders of magnitude up to a maGmum 1 OS W cm-2 was performed using calibrdted neutral filters before the sample. An analogous
336
cm2
band
set of filters after the sample made a near-null mode operation possible_ To measure the time-integrated longitudinal transmission, a laser spectroscopy detector system (Molectron) was used. Fluorescence was eliminated by cut-off filters and a large distance between sample and receiver. Fig. 1 shows the measuring result: a bleaching curve
with a plateau. Every measuring point is the mean of 10 single measurements, fig_ 1 gives a representative error width for 95% level of confidence. Checks during and after measurement did not show any measurable irreversible change of small-signal transmission_ By means of non-linear double-resonance investigations [ 121 using polarized radiation, isotropic distributions in the excited state were detected under the given measuring conditions.
3. Analytic curves
2. Experimental
between 666 and 700 nm, e.g. 0.2 < ~~~(670 nm) C 1.2 X lo-l6 “no sign of any singlet absorption above 500 nm” “a broad excited singlet absorption from 570 to 710 nm”
statements with plateau
on non-linear
transmission
If a plateau of an intensity-dependent transmission curve is defined as an area where there exist three inflection points but no extrema (this includes the interesting case that these three inflection points are far apart and transmission is nearly constant in a large environment of the central inflection point), analytic investigations show that the presence of two absorptions is necessary for the existence of a plateau and is also sufficient for suitably chosen model parameters [9]. This is true both for the case that the second ab-
Volume 100, number 4
16 September 1983
CHEMCAL PHYSICS LEIT-ERS
* .
. ...') IO7
. , .
. . ... 10'
I [w.criq Fig. 1_ X: Transmission of chlorophyll-a/dioxan solution (To = 21~5%) as a function of incidenr laser pulse (670 nmf2 ns) intensiry. o: hiafhematicai simulation of experimental curve using a model as in fig. 2c (see Jection 4).
sorption proceeds from the state the first ended in fig_ 2a and for the case that it proceeds from an energetically deeper state populated by relaxation (fig. 2b). This holds both for longitudinal and for transverse measurement. Besides it has been shown for transverse measurement in the stationary case that, by suitably chosen parameters, a plateau of any length and height can be produced and described by simple approximations. Here, the length of the plateau can be adjusted by the choice of relaxation constants, its height by the choice
of cross sections. in the non-stationary case the plateau will be shifted, in parallel to the later onset of bleaching, towards higher intensities [9]_ Last but not least analytic investigations indicate that the model necessary to describe the structure of a non-linear transmission curve is at least as large at longitudinal measurement asat transverse measurement_ (In ref. [Q] it was shown that in the stationary case, a non-linear transmission curve with longitudinal measurement can have at best as many extrema as with transverse measurement.)
4. Choice of model (energy level scheme) and numerical treatment According to section 3 the non-finear transmission of Chl-a at 670 nm is caused by a stepwise twophoton absorption according to the mode!s in fig. ?a or fig. 2b (minimum size of models). In case 3a So and S, absorptions are concerned, in case 2b So and TL absorption. (As the measuring wavelength of 670 nm lies near the maximum of So--S, absorption, on cunre
Fig. 2. Energy-level schemes for interpreting non-linear absorption of Chs-afdioxan at h = 670 nm.
347
Volume 100. rwmber 4
CHEhlIC.~l
16 September
PHYSICS LE’ITERS
its long-wave side. for singlet-singlet absorption a model of type 2b with the cycle So + S,, + S,, ++ S, can be omitted. i.e. allowance for a vibrational relaxation in St.) in case ?a. two of the four parametersare unknown: the absorption cross section US, (670) of special intetest here .md the relaxation constant kSsS, _ In case 2b. apart from the parameter ~T_~T~. all the remaining four parmnerers are known [ 13- 15]_
To determine the model effective in the given experiment and to determine the unknown excited-state parameters. the program package “Parameter identification in the spectroscopy of excited states” (PEA) 1 IO] was used *_ 1Iere. the intensity dependence of (longitudinal integral) transmission is calculated for the respective model under the given esperimental conditions - in
the first step using all known parameters and arbitrarily (in physical terms reasonably) given initial parameters for the unknown ones. Using this calculated transmission curve. an objective function valuefis determined as a measure of deviation of calculated and measured curve and this function f is then minimized in the next program step. The course of minimization shows whether the given model is sufficient and, in the confirmatory case, givesvaluesand/or limits for the unknown parameters. In the negative case (j’
does not become sufficiently small) the model has to be corrected.
Such a negative case is represented by the model 2b (So and Tt absorption) for a transmission curve according to fig. I_ (This case is particularly simple. since only one parameter is unknown_) The esperimentally found transmission course of a bleaching curve with plateau is thus caused by a two-step singlet absorption (model ?a). To determine the two
Photo”s[C&e~ lO’2 I I
Fig. 3. Standardized
population
, , ,
50” ,
.
lo” I 1 , , , ,,
1983
10=
ld6
3 1p
27
I
densities as a function of incident laser pulse (670 nm!2 ns) intensity for 4 X 10m3 molar Chl-a/ density. X: So. o: S1, 0: Tr; population
diolan solution at the cuvette end (d = 0.1 cm). A: Sa, +: S1, 0: T1; integral population density in pulse nmimum.
Volume 100. number 4
16 September 1983
CHEMICAL PHYSICS LETTERS
unknown parameters us1 (670) and ks,s,,PiSA with initial values o&O)= us and k&@s = 1011s-1 wasat first applied to model ?a and aG:ick convergence of f was reached. Then the values obtained, o&)(670) = 0.73 ~~~(670) and k!&, = 8.0X 101os-l, served as initial values in an extended model 2c, which allows for the triplet system. As expected and in accordance with the results of model 2b, there were only slight changes of parameter values in the repeated minimization process. The final parameters are 0:~(670) = 0.7 1 US (670) = 1.O X lo-l6 cm2 and kbpT = 7.6 X 101gs-r_ The respective calculated v&2 of non-linear transmission have been entered into fig. 1 for comparison with the experiment. Fig. 3 shows the intensity dependence of the main occupation densities (peak values and integrals) at the sample exit.
5. Discussion The result of a high S, absorption of Chl-a at 670 nm obtained here confirms earlier results of our group [3] and of Shepanski and Anderson [5]. The method used here avoi’ds the (one-photon) excitation of higher levels by direct S, population and thus rules out misinterpretations due to unknown deactivation paths. By applying analytic results and a numerical program package, the intensity-dependent transmission curve measured can be identified as belonging to a two-step singlet absorber and the absorption cross section uSI (670) can be determined. The present result confirms the earlier explanation of the anomalously short-wave stimulated emission of Chl-a as a consequence of S, absorption [3] and dispels doubts expressed in ref. [4]. Evidently the main cause of the discrepancy of the results of Baugher et al. [4] lies in their neglect of excited-state absorption at the pump wavelength of 337 nm. This is underlined by our experimental finding, according to which the non-linear absorption of a Chl-a solution at 337 nm shows a clear increase from 2 X 1O-5 W s cm-2 So for the evaluation of the DOD spectra excited with 2 X 1O-2 W s cm-2 [4] both knowledge of usI (337) and UT1 (337) and of the relaxation paths and constants of the states highly excited via S, +337 _ Sx , and/or T1+337 nr.r,T,, are necessary_
The high S, absorption of monomeric Chl-a in the red spectral range is an important basis for understanding the non-linear absorption and emission of chlorophyll in vivo.
Acknowledgement The authors thank Dr. P. Kis at the University of Vienna for the preparation of chlorophyll-a. The measurements described in the appendi__ were stimulated by Dr. P. Gtiber (Max-Vohner
Institute of the TU
Berlin).
Appendix.
SL absorption
in the BOB-870 range
On the basis of the abovementioned populationdensity analysis the S, absorption cross section for Chl-a/dioxan in the range 800-S70 nm was determined by an excite- and probe-beam technique. This range is of interest for instance in the investigation of the short-term kinetics of the primary redox reaction of chlorophyll in vivo. A coaxial arrangement was used for the two beams. For the excitation pulse the above dye laser was used at X = 670 run with 1, = 1.2 X 106 1%’cm-2 (at the sample input); the fluorescence of DTTC/DMSO (d= 0.2 cm; T(670 nm) = 50%) excited by the 670 nm pulse served as measuring beam. The measurement was made by monochromator (SPhl-2, Carl Zeiss Jena) by means of a multiplier and laser spectroscopy detectorsystem(Molectron). Betweenmeasuringcuvette (d = lo-’ cm; Chl-a/dioxan 4 X 10m3 mol a-l) and the entrance slit of the monochromator there was a Table 2 Sr absorption cross sections of Cl+aldiolan h (mu)
Q X 10 l6 (cm*)
800 810 820 830 840 850 860 870
0.22 0.40 0.75 1.32 0.80 0.77 1.72 1.68
349
Volume 100. number 4
CHEUICAL PHYSICS LETTERS
cut-off filter (RG 9, Schott Jena) to eliminate the 670 nxn radiation. The e~periI?lent~ll~ deterndned values of Iongitudinal integral tratwnission were corrected for the transnlission of a diosan-filled reference cuvette and the S, sbsorption cross sections calculated using the program package PEA. variant PD-2 (es&e- and probe-beam, two photon transport equations) [ 10). These are listed in table 2.
References ffuppert. P.M. Rcntzcpis and G. To&n, Biochim. Biophys. Acta 440 11976) 356. R. Arsznautt and h1.M. Denariez-Roberge. Chem. Phys. Let:ers 40 (1976) S4. D. Leupold, S Mary. P. Hoffmann, B. Hi&c and R. Ktinig,
D.
(1977) 567, J. Baughhrr. J.C. Hindman and J.J. Katz. Cbcm. RI>S
Chcll1. Phys. Letters 4s
Letters 63 (1979) 159. 3-F. Shrpanskz and R.W. Anderson Jr., Chrm. Ph!s. Letters 78 (1981) 165.
16 September 1983
161 S_ Mary, D. Leopold and J. Ehlert. to be published_ 173 M. Asano and 3-A. Ikmingstein, Chem. Phys. 57 (I 981) 1. 181 D. Leupold, 3. Ehlert and S. OberlZnder, Proceedings I. School ORLaser Pulse Fhtorometer LIF 200 (Berlin. 1982). 191 S. Oberl%nder, Analytical treatment of problems of nonlinear opkaal spectroscopy, Parts I, II. Preprint P-Math24/82 and P-Afath-O3/83 (Berlin. 1982/1983), available on request. IlO J. Ehfert and R. fahnke, PLSA pro-mm description, Parameter identification in Spectroscopy of Escited States, available on request. IllI P. Kis, Anal. Biochem. 96 (1979) 126. 1121 B. Wiesner, 8. Voigt, D. Leupold and P. Hoffmann, Proceedings IV. International Seminar on Energy Transfer in Condensed Matter (Pragttc. 1981) p. 157. rt31 H. Linschitz and I;. Sarkanen J. Am. Chem. Sot. 80 (1958) 4826. I141 G. Weber and F.W.L. Tale, J. Chem Phys. 20 (1952) 1315.
1151 A.W.H. blau and hl. Puzrt, Photocbem. Photobiol. 25 (I 977) 601.
It61 R. Jafmke and D. Leupold, Studin Biophys. 86 (1981) El.