Speotroohimioa Aota, vol. 25A, pp. 1751 to 1758. Pergsmon %#a 1969. Printed In Northern Ireland

Relation between the absorption and the emission spectra of chlorophyll a and its derivatives at room

and low temperature

J. HEVESI* and G. S. SIN~HAL~ Photosynthesis Laboratory, Department of Botany and Biophysics

University of Illinois, Urbana, Illinois, 61801

(Received 20 November 1968; revised 23 May 1909)

Abstract---Stepanov’s relation between the absorption and the emission spectra wss applied to dilute solutions (0 = 1 x 10m6M) of chlorophyll a, pheophytin a and chlorin es in EPA at 298’K and 77OK. The vibrational temperature T* of the excited molecules, calculated from this relation, depends on the frequency of the exciting light and the ambient temperature. In the system studied, T* agreed with the ambient temperature T = 298”K, in contrast to Knavtsov’s T* values of 420-630’K, proving its overall validity at room temperatures. At 77OK, the T* values were, however, found to be considerably higher (between 33’ and 133OK, depending upon the frequency of the exciting light).

The presence of two species of chlorophyll a at 77’K was most likely the reason of higher T* values. A similar AT( T* - T), observed in solutions of pheophytin a and chlorin es (where at 77’K, the second species may be present only in very small amounts), suggests that in addition to impurities, nonattainment of statistical equilibrium (due to lack of detailed balancing) of the excess vibrational energy between the excited and the non-excited molecules before emission may also cause higher values of T* at low temperatures. An increase of T* with the frequency of absorbed light was noted, which could be due to both the presence of impurities, and the increased amount of vibrational energy resulting from absorption at the shorter wave lengths.

RELATIONS between the absorption and the fluorescence spectra of complex molecules can give information regarding their shape, the frequency of pure electronic transi- tion, the validity of mirror symmetry, and the factors affecting the shape of their bands.

These relationships were studied by several workers [l, 31. STEPANOV [4, 51

proposed the following relation :

JC,(v)/E(v) = d(T) * v3 - exp * (--hv/kT) (1) where fa(r) is the fluorescence spectrum, E(V) the absorption spectrum, d(T) a con- stant independent of frequency Y, and T the absolute temperature: h and k are respectively the Planck and Boltzmann constants.

The applicability of relation (1) will require the thermal equilibrium to be estab- lished before the act of emission. As the vibrational relaxation time ( 10-11-10-12 set)

* On leave from the Institute of Experimental Physics, Attila Jozsef University, Szeged, Hungary.

t Present address: Chemistry Department, State University of New York at Albany, Albany, New York 12203.

[l] E. H. KENNARD, Phya. Rev. 11, 29 (1918). [2] E. H. KENNARD, Phys. Rev. 28, 672 (1926). [3] E. MERRIT, Phyt+. Rev. 28, 684 (1926). [4] L. P. KA~ACHENKO and B. I. STEPANOV, Opt. Spektroskopiya. 2, 339 (1957). [5] B. I. STEPANOV, Dokl. Akud. Nauk SSSR 112, 839 (1957); Soviet Phya. 2, 81 (1967).

1 1761

1752 J. HEVESI and G. S. SINUEAL

is much shorter than the emission time (IO-* see), the relation (1) should hold univers- ally so long as the system contains only one absorbing species.

ALENTSEV [S] pointed out that relation (1) can be written as:

F(v) E 3 log (Y) - log [~(~~)~~(~)] = ~~~~~~) log, + const. (2) According to (2), the plot of F(r) vs. Y (or the wsve number) should be a straight line, the slope of which is determined by the temperature of the system.

The relation (1) has been tested by several workers E&-12]. There are conflicting reports about its applic~b~ity. KETSKEMETY et al. [7] modified the relation (1) assuming that the deviation from linearity, in anti-Stokes region, is caused by decline in the fluorescence yield. As the red drop of fluorescence yield is controversial, the deviation could better be explained on the basis of inactive absorption caused by the presence of some nonfluorescing impurities.

In some studies [9, lo], the temperature (!Z’*) determined from the slope of the straight line of relation (1) is higher than the ambient temperature (T). Presence of an impurity is generally the cause given for higher T*.

The first application of relation (1) to systems of biological interest-solutions of chlorophyll a and b was made by KRAVTSOV [ 131. He found the temperature T*, calculated from the slope of the P(Y) plot, to be much higher than the ambient temper&t~e, T. Recently, SZALAY et al. [14] applied relation f 1) to different algae, as well as to chlorophyll a solutions. They assumed that light-absorbing impurities (such as a non-fluorescent form of chlorophyll a) were responsible for deviations of P(Y) plots from linearity.

The aim of the present work was to check the app~c&bility of relation (1) to substances of biological interests, such as chlorophyll a, pheophytiu CE and chloriu e8 and to study the influence of the ambient temperature and of the frequency of the exciting light, on the vibrational temperature of the excited molecules.

EXPERIMENTAL

Preparation of chlorophyll a, pheophytin cz and chlorin e, is described in Ref. [15]. Absorption spectra were measured with a Bausch and Lomb Spectronic 505 optical density recording spectrophotome~r. Fluorescence spectra were measured with an instrument assembled in this laboratory 1161. Fluorescence was measured on the

[6] M. N. ALENTSXV, Opt. S~ek~roskop~~~. 4, 690 (1958). 1’71 I. KETSKEXETY, J. DOXBI and R. HORVAI, Acta Phya. Hang. 12, 263 (1960); Am. Phy8.

7, 342 (1961); dcta Phys. Hung. 14, 165 (1902). [8] N. A. BORISEVICH, V. V. GRUZINSKII and V. A. TOLKACHEV, Opt. Spectq 16, 94 (1964). [Cl] M. N. ALENTSEV and L. A. PAHOMITCHEVA, IAN Phys. Ser. SSSR 24, 734 (1960); Ball.

Acad. S&i. P&ye. Ser. SSSR 24, 737 (19601. [lo] Yu. T. MAZ~RENKO and B. S. NEPORENT, Opt. Speetry 12, 317 (1962). [ll] L. A. KRA~SOV and A. N. RWINOV, Opt. Spectry 12,355 (1962). [12] J. HEVESI ctnd L. KOZMA, dcta Phya. Chem. Szeged 8, 103 (1962). [133 L. A. KRA~SOV, Engng Phys. J. 2, 45 (1968); Bull. Acad. Sci. Phys. Ser. SSSR 28, 77

(1959). [14] L. SWAY, E. RABINO~ITCH, N. R. MARTY and GOVIND~E, Biophy& J. 7, 137 (1967). [15] G. S. SINUHAL, W. P. WILLIAMS and E. RABINOWITCH, J. Phys. Chem. 72, 3941 (1968). Cl63 N. R. MURTY and D. FRACKOWIAK, Cwrrents in Photoqnthtmk (Edited by J. B. THOMAS and

J. H. C. GOEDHEIZR), p. 9. Ad. Danker (1966).

Absorption and emission of chlorophyll a at room and low temperature 1753

light-exposed side of the cell. The intensity of the exciting light was reduced by means of neutral density filters, whenever the fluorescence signal was so strong that the photomultiplier (RCA C7268 with S-20 response) moved into the non-linear range. To remove the stray incident light, a CS 2-58 Corning glass red cut-off filter was used. Fluorescence spectra were corrected for photomultiplier response. For low temperature measurement the sample cuvette was suspended in a Dewar flask which was filled with liquid nitrogen. Other experimental details about absorption and fluorescence measurements are given in Ref. [ 151. All the solvents were reagent grade ; they were used without further purification. In absorption measurements, the solute concentrations were between 1 and 2 x 10-6M; in fluorescence measure- ments 1 x lo-” M.

RESULTS AND DISCUSSION

Figure 1 shows the absorption and the fluorescence spectra, and the function P(V) (in arbitrary units) for solutions of chlorophyll a (A), pheophytin a (B) and chlorin e, (C), in EPA (ether + isopentane + ethyl alcohol), vs. wave number, +, at 298°K. The wave length of the exciting light was near the blue absorption maximum. The function F(5) is seen to be linear over a wide range for all the three substances, but a deviation from linearity appears at frequencies near the far red end of the absorption band. According to KETSKEMETY et al. [7], this deviation is due to a “red drop” in the fluorescence quantum yield. Inactive absorption, in the region of non-linearity, is supposed to be the cause for this drop in the quantum yield. Though the existence of the red drop in dilute solutions is controversial, a deviation of F(f) function from linearity (cf. Fig. 1) may suggest the existence of a red drop of quantum yield in solutions. We are, however, not certain of the origin of this deviation.

The absorption peaks, il,, (max.) and the fluorescence peaks, A,,,, (max.), the wave lengths of pure electron transitions, A,,, and the temperature T* (calculated from the slopes of the F”(F) plots at 298’K) are given in Table 1. In all cases, T* is in fair agreement (&30”K) with the ambient temperature T. This observation is in contradiction with that of KRAVTSOV [ 131, who found much higher values of T* (420-630°K) for chlorophyll a, and pheophytin a, having concentration of about 1 x 1O-6 M. There is no correlation between the differences 1, - A,,, and T* - T.

In Fig. 2, the function F(c) is plotted versus wave number, Y, for four different excitation frequencies for solutions of chlorin e6 at 298°K. Over a wide range, P(S) is linear in all the cases. The value of T* for excitation at 390, 400, 405 and 410 nm are respectively 327.7’, 301.5”, 288.7” and 283.5”K (cf. Fig. 2). There is correlation between il,, - A,,, and T* - T. Parallelism between 1, - &,, and T* - T could be expected, if vibrational energy acquired in the absorption act was the origin of the excess vibrational temperature (T* - T) at the time of emission.

Figure 3 contains the plots of F(G) vs. f and values of T* plotted as function of the wave length iz of the exciting light for chlorophyll a at 77°K. Again, P(G) is linear over a wide range ; fluorescence peak of chlorophyll a solutions depends, at 77’K, upon the wavelength of excitation. (The peak positions are given in Table 2, for all three compounds studied.)

The values of T*, found at 77”K, show a strong dependence upon the wavelength of the exciting light (cf. Table 2). While T* values were in fair agreement with the

1754

too

0

J. HEVESI and G. S. SINGHAL

1.45 I.50 1.55

p x IO-4cm-1

Fig. 1. Absorption and fluorescence spectra and P(c) vs. wave number (i) in EPA

solutions at 298%. (A) for chlorophyll a at 430 nm excitation. (B) for pheophytin a at 410 nm excitation. (C) for chlorin e, at 390 nm excitation.

Table 1. Spectral properties and the local temperature of pheophytin a, chlorophyll a and chlorin es in EPA (ether + isopentane + ethyl alcohol)

at room temperature

Fluorescent substances

Pheophytin a Chlorophyll a Chlorin es

410 430 390

Aabs(maxa) J,,(max.) 10 20 - &XC (nm) (nm) (nm) (nm) (Z)

666.0 676.0 671.5 261.5 277.4 661.5 672.0 668.0 238.0 294.8 663.0 669.0 665.7 275.7 327.9

6.0

5.0

c’ u) E

y 4.0

is iY

3.0

20

I.0

I I I I I I I I I I 45 150 I .55 I 45 I .50 1.55

Ix 10m4cm-b ii x 10e4 cm-l

Fig. 2. F(f) vs. wave number (9) for solu- Fig. 3. P(f) vs. wave number (G) for solution of tion of chlorin es at 298’K; curves 1, 2, 3 chlorophyll a at 77’K; curves 1,2,3 and 4 refer and 4 refer to excitation at 390, 400, 405 to excitation at 435, 440, 445 and 456 run re-

and 410 nm respectively. Insert shows spectively. Insert shows calculated T* vs. wave calculated T* vs. wave length (A). length (A).

Table 2. Spectral properties and local temperatures of chlorin e,, chlorophyll a and pheophytin a in EPA (ether + isopentsne + ethyl alcohol) excited by different

wave lengths at 77°K.

Fluorescent substances

&&nax*) &,(max.) 10 IO - 4?x,

(nm) (nm) (nm) km) (G)

/ 436 608

Chlorophyll a I

440 668 445 668

t 455 668 405 606

Pheophytin a L 420 666 430 665

/ 390 662 Chlorin 400 662

es 410 662 420 662

672.0 670.0 235.0 160.9 672.5 670.3 230.6 129.1 674.0 671.0 226.0 119.1 678.5 673.7 218.7 110.2 669.0 667.0 262.0 176.8 670.0 667.5 247.0 160.3 671.0 668.0 238.0 160.0 666.0 663.5 273.6 206.0 665.6 663.7 263.7 199.1 666.0 664.0 254.0 186.2 667.0 664.5 244.5 172.3

1755

1756

4.0

3.0

i I% s +

iz LL

2.0

J. HIEVESI and G. S. SINQEAL

I ’ ’ ’ ’ ’ ’ ’ ’ /

A

- 170

‘K

150

1.45 1.50 1.55

F x lO+cm-1

Fig. 4. .F(ti) vs. wave number (v) for solution of pheophytin a at 77’K; curves 1, 2 and 3 refer to excitation at 390, 420 and 430 nm respectively. Insert shows

calculated T* vs. wave length (A).

ambient temperature at room temperature (298’K), a considerable difference (33-12gaK, depending upon the frequency of the exciting light) between T+ and T appears at 77’K for all three compounds. The dissipation of excess vibrational energy of excited molecules thus appears- not implausibly-to be slowed down at low temperatures.

Figure 4 shows the plots of J’(Y) vs. wave number, +, at three different wavelengths of excitation for solutions of pheophytin a at WK, and the calculated values of T* as a function of the wavelength of excitation. The linear relation is applicable in

this case too, over a wide range of t values. The calculated values of T*, in this

case, change linearly, with A. The vibrational temperature T* for pheophytin a also is higher than the ambient temperature T.

Figure 5 gives plots of F(-) v vs. wave number, 5, and of T* as function of the

Absorption and emission of chlorophyll a at room and low temperature 1757

4.0

3.0

c’ r 8 + 6 L

2.0

I .o

I I 1 I a I I I I

\

a

390 400 410 420

1.45 1.50 1.55

I x 10-4cm-I Fig. 5. P(C) vs. wave number (9) for solution of chlorin e6 at 77’K; curvea 1, 2, 3 and 4 refer to excitation at 390, 400, 410 and 420 nm respectively. Insert shows

calculated T* VS. wave length (A).

wavelength of excitation for solution of chlorin e6 at 77°K. The values of T*, in this case too, are much higher than the ambient temperature (77°K).

Presence of two species or of some impurity and the lack of Boltzmann distribution can possibly be the two reasons which could explain the observed higher values of T* (cf. Table 2). It has been shown by ALENTSEV and PAKHAMYCHEVO [9], and by KRAVTSOV and RUBINOV [l l] that presence of impurities can cause an increase in the value of T*. However, according to the observations of the latter authors, the proportion of impurities present in the system should be quite considerable (about 50 %) to have a significant difference in T* values.

The insignificant difference between T* and T at 29VK (Table 1) suggests an absence of impurities or of any other species at room temperature. A sign&ant difference between T* and T at 77°K may, however, suggest the presence of other species at liquid nitrogen temperature. On the basis of the magnitude of AT (T* - T), we can conclude thaf at 77”K, the proportion of impurities in the solutions

1758 J. HEVESI and G. S. SIN~HAL

of all the three substances should be of the same degree. On the other hand, SIN~HAL et al. [15] using matrix analysis, found that at 77”K, while two species of chlorophyll a (mono- and disolvates) are present in significant proportions, practically no or very small amount of a second species is present in case of pheophytm a or chlorin e6. This suggests that probably the other factor (i.e., lack of statistical equilibrium over vibrational levels of excited and non-excited molecules), besides impurities, may also cause an increase in T* values. The dependency of T* on the frequency of excitation may also support this suggestion. Though the vibrational relaxation process, even at very low temperatures, is supposed to be completed in a time of the order of lo-l1 set, there could possibly exist a situation in the frozen solutions at 77”K, where the principle of detailed balancing [18], at the time of emission, is not satisfied. A similar conclusion was also reached by KLOCHKOV and KOVOTKOV [ 171.

We therefore, conclude that the main cause of higher T* values at 77’K is prob- ably the presence of other species. However, the data for pheophytin a and chlorin e6 suggest that the higher T* values could also be caused by non-establishment of statistical equilibrium, before the act of emission, over the vibrational levels of excited and non-excited molecules.

Acknowledgmtmts-We wish to thank Professor E. RAEZINOWITCH for a very fruitful discussion during the course of this investigation, and Professor GOVINDJEE for helpful suggestions.

Financial support from National Science Foundation and Atomic Energy Commission is gratefully acknowledged.

[17] V. P. KLOCHKOV and S. M. KOROTKOV, Opt. Spectry 22, 189 (1967). [18] W. HEITLER, Quantum Theory of Rudiatim, p. 412. Clarendon Press (1954).