A study of the fluorescence quenching of chlorophyll by photoacoustic spectrometry

Spectrochmtca

Acm, Vol 4lA,

No

6, pp 833-836

1985

F’nnted m Great Bntam

0

A study of the fluorescence quenching of chlorophyll photoacoustic spectrometry

0584-8539/85 S3 00 + 0 00 1985 Pergamon PressLtd

by

V UPADHYAYA, L B TIWARI* and S P MIsHRAt Department of Chemistry, Faculty of Science, Banaras Hindu Unlverslty, Varanasl 221005, India (Recetvedfor publlcarlon 2 Januar) 1985) Abstract-The fluorescence quenching of chlorophyll a m acetone by chloraml, p-benzoqumone and mduutrobenzene has been studied using a single beam photoacoustlc spectrometer Using a modified Stern-Volmer equation the quenchmg constants have also been determined

with a quartz cell of 1 cm optical path length The concentratlon of chlorophyll was calculated usmg the specific absorption coefficient (82 04 g - 1cm- ’ at 665 mp) quoted by MYERS and KRATZ [12] The quenchers chloranll, mduutrobenzene and freshly prepared p-benzoqmnone [ 133 were recrystalhzed from benzene, rectified spirit and petroleum ether, respectively Acetone was redistilled by the usual procedure before use The photoacoustlc spectra of chlorophyll were recorded on an mdlgenously fabricated single beam photoacoustlc spectrometer [ 1l] The source of radlatlon was a 600W Phlhps tungsten halogen lamp in conJunctlon with a CEL 0 25 grating monochromator The photoacoustlc spectrum of the sample was normahzed using the power spectrum of the source The choppmg frequency was fixed at 22 Hz to maxlmlze the signal/noise ratlo and the depth of the sample cell was 2 mm Different concentrations of the quencher solutions were prepared m acetone To prepare chlorophyllyuencher solutions 10 ml of the prepared stock of chlorophyll was delivered volumetrically to previously weIghed samples In this manner the mltlal chlorophyll concentration was kept constant for all solutions

INTRODUCTION The fluorescence of chlorophyll, because of its lmportante m understanding the mechanism of photosynthesis, has been the subject of extensive mvestigatlons

during the past halfcentury [l-5] It 1salso known that the fluorescence of chlorophyll m solution 1squenched efficiently by various oxldlzmg agents and moderately by certain aromatic reducing agents [M] Recently a new spectroscopic techmque has been developed and used for momtormg non-radiative deexcitations that occur m solid or hquld samples followmg their excltatlon by the absorption of photons [9, lo] In this novel technique, called photoacoustic spectrometry (PAS), the intensity modulated (chopped) monochromatic beam of light 1s incident upon the sample material enclosed m a cell of constant volume If the sample absorbs at the wavelength of incident radiation, on subsequent de-exntatlon, the absorbed energy may appear as heat and cause a perlodlc pressure variation m the gas surrounding the sample This pressure variation occurs at the intensity modulation frequency and 1s detected by a sensitive microphone transducer Smce the physlcal prmclple of photoacoustic spectrometry 1sbased on the radlatlonless conversion of the absorbed energy for the production of an acoustic signal, It is complementary to conventional luminescence spectroscopy In the present commumcatlon we have used a single beam photoacoustic spectrometer [ 1l] to study the quenching of the fluorescence of chlorophyll by chloraml, p-benzoqumone and m-dmltrobenzene The quenching constants (Stern-Volmer constants) have been determined by a modified Stern-Volmer equation

RESULTS AND DISCUSSION The optical absorption and the PAS spectra of chlorophyll a m acetone are shown m Fig 1 The peaks at 660 and 430 nm m the PAS spectra are the characteristic absorptlons of chlorophyll As shown m Fig 2 the PAS slgnal increases linearly with the concentration of chlorophyll at a fixed excitation wavelength This indicates that m the concentration range used m our experiments mterfermg effects due to optical and thermal saturation are not present [lo] The varlatlon of the PAS signal of chlorophyll at 660 nm m the presence ofdlfferent concentrations ofpbenzoqumone 1s shown m Fig 3 An increase m the PAS signal of chlorophyll 1s observed on addition of the quencher solutions (cf Table 1) Since the PAS signals of all the quenchers, m acetone, at given concentrations are not detectable at 660 nm, the additional PAS signals obviously may result from the fluorescence quenching of chlorophyll The PAS signal increases with the concentration of the quencher solutions and attams a hmltmg value for all the samples studled This hmlt corresponds to complete quenching of fluorescence, since the existence of an “unquenchable fluorescence” as predicted by FRANK

EXPERIMENTAL Chlorophyll a was extracted m acetone from the blue green alga Nostoc c&zcola [12] and Its optical absorption spectra were recorded on a Beckman Model 35 spectrophotometer

*Present address Department of Physics, Banaras Hindu University, Varanasi 221005, India tAuthor to whom correspondence should be addressed

and LIVINGSTON [7] has not been proven expenmentally[8] If the thermal and elastic properties of the 833

V

UPADHYAYA

et al

(b)

Wavelength

(n m)

Fig 1 (a) PAS spectra of chlorophyll III acetone (b) Optical absorption spectra of chlorophyll m acetone

I 0 002

0 OOL

0006

5

I

I 500

CONCENTRATION CONCENTRATION

OF CHLOROPHYL.

I

I

1000

OF p- BENZOPUINONE

Cmma,I-’ 1

[ m mot t-’ 1

Fig 2 Vanatlon of PAS signal (at 660nm) with concentration of chlorophyll m acetone

Fig 3 Effect of pbenzoqumone concentration on the PAS slgnal ofa solution of4 0 x 10e6 M chlorophyll m acetone (at 660 nm)

Fluorescence quenchmg of chlorophyll

835

Table 1 Stern-Volmer constants for chlorophyll a in acetone at 25°C PAS signal for chlorophyll solution (&PAS),) = 1 5 at 660 nm

Quencher

Concentration of the quencher (mol I-‘)

Chloraml

0002 0006

p-Benzoqumone

m-Duutrobenzene

PAS signal of the chlorophyllquencher solution at 660 nm

0010

1 54 1 58 160

0005

1 56

0015 0 025

160

0005 0 025 0040

1 54 160 I 62

IO

l+k[Q]

-

where k 1s the quenching (Stern-Volmer) constant and I, 1s the intensity of the unquenched fluorescence under the condltlons of the experiment If the production of new chemical species through the absorption of photons does not occur under experimental con-

4(PAS,- 4(PAs), fraction ~U’As),

(fraction c

'

vo

IO

= c

$

(IO-I)

(3)

where 4(PASj1s the PAS signal of chlorophyll a m the presence of the quencher molecule From Eqns (l), (2) and (3) we have

&=klQl decaying

non-radlatlvely

decaying

radlatlvely

A plot of p/1 -p vs the quencher concentrations (Fig 4) gives straight hnes mdlcatmg that the mod&ications of the Stern-Volmer equation suggested by ROLLEFSON and BOAS [17] and LIVINGSTON and CHUN LIN KE[~] need not be applied The k values obtained from the slopes of the straight hnes (cf Fig 4) are given m Table 1 It has already been shown that the observed quenchmg IS not due to dlffuslonal and static processes alone and depends on other properties of the solvent [18] A charge-transfer mteractton may be possible between chlorophyll a and the quencher molecules The cyclopentanone region (rmg V) of the chlorophyll molecule

of the excited molecule

(fraction

63+3

4 (PAS)-&PAS),

fraction

dmons, the radiative decay accompanied by the emlsslon of lummescence (fluorescence or phosphorescence) and the non-radiative de-exatatlon with full converslon of the absorbed energy mto heat are the only de-exatatlon modes [ 151 Phosphorescence IS not observed at room temperature because competltlve non-radlatlve processes such as lmpunty quenching or Internal conversion to the singlet ground state are operative Therefore, the intensity of the unquenched fluorescence would be proportional to the fraction of the optically excited molecules decaying radlatlvely The PAS signal 4(PASj, of the chlorophyll molecule alone depends linearly on the quantum efficiency of non-radiative de-exatatlon [ 161, thus

4 (PAS),=

llOf5

where v. 1s the frequency of the exciting radiation and vJ 1s the mean frequency of the fluorescent radlatlon, the term v//v0 1s a correction factor introduced to account for the red shift of fluorescence and C 1s a constant depending on unknown variables The quenchmg of fluorescence, which 1s caused by the addltlon of quencher to a solution of chlorophyll, increases the conversion efficiency of the absorbed energy mto heat by a non-radiative mechanism If the deviation m the PAS slgnal IS only due to the quenchmg of fluorescence, the quenched lummescent energy, I, - I, as a result of the addition of the quencher directly, gives the additional PAS signal at the excitation wavelength Thus

(1)

If/l =

16Ok5

162

molecule do not change as a result of interaction with the quencher molecules, the ratlo of the unquenched PAS signal (of a solution of chlorophyll alone) and the completely quenched PAS signal gives the fraction of the optically excited molecules decaying nonradlatlvely [14] For chlorophyll a this ratlo 1sfound to be 0 9, mdlcatmg that 90 % of the excited molecules are decaymg non-radlatlvely (Fig 3), which IS m good agreement with the previously reported value of 10 % quantum yield of fluorescence [l] The varlatlon of Intensity I of the fluorescence as a function of the concentration [Q] of the quencher, for low concentration values, corresponds to the well known Stern-Volmer equation r=

k

(I mol-‘)

@(PAS,)

decaying

of the excited molecule

non-radlatlvely)

decaymg

radlatlvely)

(2)

836

V UPADHYAYAet al

2

'I

.~ /

/

•

I

I 20

p- BENZO(;IUINONE m-DINITROBENZENE

~

i

I ~,O

l

CONCENTRATION OF GLUENCHER [ m mot i,-I]

Fig 4 Variation of/z/1 - / z vs quencher concentration

is a centre of high electron density [19] and all the quencher molecules taken are well known electron acceptors [20] The k values obtained are m the order of decreasing acceptor strength of the quencher molecules VlZ chlorand > p-benzoqumone > mdmitrobenzene [20] The possible formation of a nonfluorescent ground state molecular complex as a result of addition of the quencher to the fluorescer molecule will lead to a quenching of the fluorescence l21] Acknowledgements--The authors are thankful to Professor S N THAKUR, Department of Physics, Banaras Hindu University for valuable discussions, critically reading the manuscript and providing laboratory facilities for PAS measurements They are also thankful to Dr A K PANDEY, Department of Botany, Banaras Hindu University for the extraction of chlorophyll used in the experiment One of us (V U ) is thankful to the University Grants Commission, New Delhi for the award of a Teacher Research Fellowship

REFERENCES

[1] T A PRINS, Nature, Lond 134, 457 (1934) [2] J FRANCKand R W WOOD, dr chem Phys 4, 551 (1936) [.3] J FRANCK,C FRENCHand T PucK, J phys Chem 45, 1268 (1941) [4] M CALVIN,J theoret Blol 1, 258 (1961)

['5] V B EVSTIGNEEV,Photochem Photobzol 4, 171 (1965) [-6] J FRANCKand H LEVI, Z Physlk Chem B27, 409 (1934) [7] J FRANCKand R LIVINGSTON,J chem Phys 9, 184 (1941) [8] R LIVINGSTONand CHUNL1N KE, J Am chem Soc 72, 909 (1950) [.9] A ROSENCWAIG,Adv Electronics Electron Phys 46, 242 (1978) [10] M J ADAMS,J G HIGHFIELDSandG F KIRKBRIGHT, Analyt Chem 49, 12 (1977) [11] V N RAI, L B TIWARI,S N THAKURand D K RAI, Pramana 19(6), 579 (1982) [12] J MYERSand W A KRATZ, J Ken Phys~ol 39, 11 (1955) [13] A VOGEL, Practical Oroanw Chemistry Longman, London (1978) [14] P GANGULYand C N R RAO, Proc Ind Acad Sci (Chem Sci) 90(3), 194 (1981) [,15] A MANDELIS,Chem Phys Letts 91(6), 501 (1982) [16] A ROSENCWAIGand A GERSHO,J appl Phys 47, 64 (1976) [17] G ROLLEFSONandH BOAS,J Phys Coil Chem 52,518 (1948) [18] G K HODGESandV LAMER,J Am chem Soc 70,722 (1948) [19] J R LARRYand Q VANWINCKLE,J phys Chem 73(3), 570 (1969) [20] R FOSTER, Organic Charge Transfer Complexes Acadenuc Press, London (1969) [21] M A SLIFKIN, Charge Transfer Interaction of Biomolecules Academic Press, London (1971)