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Hydrated chlorophyll a oligomers in solutions, monolayers, and thin films
Journal of Molecular Structure, 297 (1993) l- 11 0022-2860/93/$06.00 0 1993 - Elsevier Science Publishers B.V. All rights reserved
Hydrated chlorophyll a oligomers in solutions, monolayers, and thin films Bogumil Zelent, Judith Gallant, Alexander G. Volkov’, Maya I. Gugeshashvili2, Gaktan Munger, Heidar-Ali Tajmir-Riahi, Roger M. Leblanc* Centre de recherche en photobiophysique, Universite’ du Qukbec h Trois-Rivi&es, 3351, boul. des Forges, C.P. 500, Trois-Rivieres, Que. G9A 5H7, Canada (Received 2 November 1992) Abstract The interfacial tension measurements of dry and wet chlorophyll a at an octane-water interface show differences in adsorption behavior of the pigment depending on its aggregation state. Optical properties were also investigated using absorption and Fourier transform-infrared spectroscopy, along with fluorescence (excitation and emission) measurements in non-polar solvents and in thin films. The absorption and fluorescence spectral characteristics, and fluorescence lifetime measurements of dry and wet samples clearly indicate the difference in aggregation properties of this photosynthetic pigment. A strong decrease in the energy of QY electronic transitions (Av = 1700cm-‘), due to the formation of hydrated oligomers, was accompanied by a decrease in fluorescence lifetime from 5.75 f 0.02ns (diluted dry chlorophyll a in n-hexane) to 0.12 f 0.03 ns (wet chlorophyll a in thin films). Discussion of these results is based on the key role of the central magnesium atom in the porphyrin ring in intermolecular interactions and self-organization of chlorophyll a molecules.
Introduction The electronic charge distribution of the r-electron conjugated system in the porphyrin nuclear framework of chlorophyll a (chl a) is highly sensitive to environmental interactions. This is manifested by the energy levels and the probability of electronic transitions from the ground to the singlet excited electronic states which are polarized along the x and y axes of the porphyrin ring [1,2]. The changes in the electronic absorption spectra of chl a caused by intermolecular interactions like chl a-chl a or chl a-water [3-71 and chl a-protein [8] in non*Corresponding author. Tel: (819) 376-5077.Fax: (819) 376-5057.E-mail:[email protected] CA. ’ Dedicated to Professor CamilleSandorfy. ‘Permanent address: The A.N. Frumkin Institute of Electrochemistry,Acad. Sci. USSR, 31 Leninsky Prospekt, Moscow 117071,Russian Federation.
polar and polar solvents have been widely studied in view of the numerous roles played by chl a in the primary events of plant photosynthesis: light harvesting, energy transfer and charge separation in photosynthetic units [g-17]. The state of chl a molecules in green leaves is believed to be in different aggregated forms and is still under investigation. The interface between two immiscible liquids with immobilized photosynthetic pigments can serve as the simplest model of a biological membrane convenient for the investigation of photoprocesses that are accompanied by spatial separation of charges. It was demonstrated by mass spectroscopy and polarographic studies [16] that on illumination of wet chl a at an octane-water interface, oxygen evolution with a quantum efficiency of lo20% takes place in the presence of proton acceptors (pentachlorophenol or 2,4-dinitrophenol) in
2
octane and electron acceptors (nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide phosphate (NADP+) or K3[Fe(CN)6]) in water. This process is accompanied by the appearance of a photopotential at the octane-water interface. The process occurs at the interface and the photopotential and reaction rate are proportional to the surface concentration of chl a. However, optically transparent electrodes, modified with monolayers of photosynthetic pigments using the Langmuir-Blodgett (LB) technique, are also suitable for the investigation of chemical models of photosynthesis [17]. During photocatalytic oxidation of hydroquinone by chl a or pheophytin a in monolayers, the standard Gibbs free energy of electrochemical reaction is negative and hence solar energy was used to overcome the activation barrier. In the absence of hydroquinone, it is possible to use water molecules as electron donors and NADP+ or methyl viologen as an electron acceptor. In this case solar energy can be used to perform work, since the standard Gibbs free energy of the reaction of water photooxidation and NADP+ reduction is positive. This process of artificial photosynthesis is the chemical model of natural photosynthesis. The utilization of solar energy for the transport of electrons from water to NADP+ is the most important process in the bioenergetics of natural photosynthesis. Considering the importance of chemical models of photosynthesis, in this paper we have investigated the interfacial tension at the octane-water interface as well as the optical properties of dry and wet chl a. Some new spectroscopic results for dry and wet samples in solutions and in thin films are presented and discussed with regard to their different structural organization.
Experimental Samples and reagents
Chl a was purchased from Fluka Chemical Corp. (Ronkonkoma, NY) in a 1 mg ampoule, and
B. Zelent et al.lJ. Mol. Struct., 297 (1993) l-11
arachidic acid from Applied Science Laboratories Inc. (State College, PA). The deionized water (Nanopure filter system, Barnsted, Boston, MA) used to prepare all the solutions was doubly distilled in quartz (Model Bi 18, HeraeusQuarzschmelze, Hanau, Germany). Its specific resistivity was 18 MRcm, and its surface tension was higher than 71 mN m-l. Benzene from Anachemia Accusolv (Montreal, Que.), used to produce dry chl a solutions, was distilled at 80°C in a 3Ocm Vigreux column. nHexane was purchased from Aldrich (Milwaukee, WI), and chloroform from Anachemia Accusolv (Montreal, Que.). They were both distilled before use. n-Octane was bought from BDH Chemicals Canada Ltd. (Toronto, Ont.) and Fluka Chemie AG (Buchs, Switzerland). The Fourier transform-infrared (FT-IR) spectra of the hydrated chl a oligomers were measured in thin films. The substrate was a 35 x 9 x 2mm CaF2 crystal. Its cleaning procedure included soaking in a phosphate free, non-ionic, neutral pH liquid Aquet detergent, purchased from Manostat Laboratory Instruments and Scientific Apparatus (New York), followed by rinsing in chloroform and in methanol. All these steps were carried out in an ultrasonic bath which was regularly filled with ice to prevent overheating since CaF2 is sensitive to thermal shock. All experiments were performed at a temperature of 20°C under dim green light.
Hydrated chlorophyll a oligomer preparation
Wet n-hexane (or n-octane) was prepared with equal volumes of the solvent and water equilibrated for 48 h. After equilibration the solvent was transferred to a vial containing chl a and was sonicated for 100min in a cooled ultrasonic bath. During sonication, the formation of hydrated oligomer was controlled by UV/visible spectroscopy. The usual concentration of hydrated chl a solutions was 2 x 10m5M.
3
B. Zelent et al./J. Mol. Struct.. 297 (1993) I-11
Interfacial tension measurements The interfacial tension at the octane-water interface was measured by determining the weight and volume of the drop falling from the end of a capillary tube under its own gravity. The surface tension (y) was obtained from Eq. (1) Y = Vdl
(1)
- d2)Wr
where d, and d2 are densities of the liquid studied, V the drop volume, g the acceleration of gravity, F a correction function found in the Harkins-Broun table, and I the capillary radius. The equipment used to measure the interfacial tension consisted of a glass capillary, a 1 cm3 syringe, a micrometer and a vessel filled by a liquid with lower density used to immerse the capillary. The internal capillary diameter was 0.6 mm. The drop lifetime necessary to establish adsorption equilibrium was not less than 4min.
Langmuir-BlodgettJ
deposition
In the case of some FT-IR measurements, multilayers of cadmium (Cd) arachidate were deposited on the freshly cleaned CaF2 substrate. A total of ten monolayers were deposited (five on each side). The Langmuir trough was described elsewhere [ 181. The subphase used to spread arachidic acid was a 10e3 M tris(hydroxymethyl)aminomethane Trizma base buffer set at pH 6.5, with 10T4M cadmium chloride. Spreading solvent was freshly distilled degassed chloroform. Room and subphase temperatures were maintained at 20°C during deposition. The surface pressure for LB deposition was set at 30mNm-‘.
Preparation of thin jilms Thin films of hydrated chl a oligomers were prepared for electronic absorption and fluorescence spectroscopies. The quartz slides were dipped in the solution of hydrated oligomers which were
easily adsorbed onto the surface and dried with a slow stream of argon. Thin films of hydrated oligomers were also investigated by FT-IR spectroscopy. They were prepared by a solvent evaporation technique. The hydrated oligomer solutions were directly deposited on the surface of a Cd arachidate covered or a freshly cleaned CaF2 crystal. Each drop was dried with a flow of argon or nitrogen before adding some new material to the surface to prevent its oxidation.
Photophysical measurements Absorption spectra were performed from 350 to 800nm with a Hewlett-Packard diode array spectrophotometer, model 8452A (Palo Alto, CA), controlled by an IBM personal system/2 computer. In the case of hydrated oligomer thin films deposited on CaF2 (those used for FT-IR spectroscopy), the absorption reference spectra of the Cd arachidate or the cleaned CaF2 substrate were subtracted from the absorption spectra. The fluorescence emission and excitation spectra of dry and wet chl a solutions and thin films on quartz slides were registered by means of a Spex Fluorolog II spectrofluorimeter, model 1870 (Spex Industries Inc., Metuchen, NJ), equipped with a Datamate DM 1 data acquisition system and a thermoelectrically cooled Hamamatsu, model R928 photomultiplier tube (Hamamatsu Photonic Systems Corp., Bridgewater, NJ). The spectra were corrected for the lamp intensity and for the photomultiplier sensitivity. The fluorescence lifetimes of the same samples were measured using Photochemical Research Associates International Inc. Model 3000 fluorescence lifetime, single photon counting equipment (PRA, London, Ont.). The excitation light was generated by nitrogen flash lamp PRA model 510. The samples were illuminated in the spectral region of 440 f 50 nm using a bandpass filter (Oriel, Stratford, CT). Fluorescence emission was detected at 670 f 5 nm or 750 f 5 nm using bandpass interfer-
B. Zelent et al./J. Mol. Strut.,
0
’
I
1
100
200
I
AREA
Fig. 1. The dependence
of surface pressure at the octane-water
ence filters (Ditric Optics Inc., Hudson, MA). Data were analyzed by PRA statistical deconvolution program. The infrared (IR) spectra were recorded on a Bomem DA3.02 FT-IR spectrophotometer (Vanier, Que.), equipped with a liquid nitrogen cooled HgCdTe detector. A total of 500 scans was carried out with a resolution of 4cm-‘. Baseline correction and smoothing procedure were also
I
300
297 (1993)
I-11
I
400
500
(A*)
interface on surface concentration
of dry (1) and wet chl a (2).
performed. The reference spectra were recorded with Cd arachidate covered or with cleaned CaFz crystals depending on whether or not the sample contained the Cd arachidate layers. Results and discussion Chl a molecules are known to have an abundant surface activity and therefore, they considerably
LOS-
7457::
A
I i: ,
0.64.
400
500 600 WAVELENGTH
Fig. 2. Electronic absorption
700 (nm)
6f
400
500 600 WAVELENGTH,
700
601
spectra of (A) wet (1) and dry chl a (2) in n-hexane solution and (B) wet chl a in thin films on a quartz slide.
5
B. Zelent et al./J. Mol. Struct., 297 (1993) 1-11
reduce the tension during adsorption at the interface This property was used to determine the surface excess of chl a according to Gibbs theory. Figure 1 shows the dependence of interfacial tension on the chl a concentration in dry (curve 1) and wet (curve 2) n-octane. A gradual reduction of y can be observed in the chl a concentration ranges from 1 x 10e6 to 5 x 10m6M for wet and from 1 x low6 to 1.5 x lo-’ M for dry solutions. For the dry solution, the standard free Gibbs energy of adsorption equilibrium is equal to -27.4 kJ mol-’ . For hydrated oligomers, the standard free Gibbs energy is equal to -35.2 kJ mol-‘. The positive attraction constant a = 0.59 for dry chl a shows the attraction interaction between the adsorbed molecules. The negative constant
a = -0.215 indicated repulsion between clusters of hydrated oligomers. The difference between the two curves is in the hydration state of the chl a solutions and the structural organization of the adsorbed particles. This is a rare example in colloid chemistry where a surface active compound can be found in two different adsorbed states, as dry monomeric molecules and as self-organized hydrated molecular clusters. The investigations on biologically active compounds from membranes of chloroplasts, mitochondria and bacteria led us to study a new type of surface active compound, capable of forming self-organized molecular assemblies. When chl a is dissolved in the water-saturated hydrocarbon, an additional band appears in the
A
I
WAVELENGTH (nm)
WAVELENGTH ( nm )
WAVELENGTH
(nm 1
Fig. 3. Corrected fluorescence spectra of (A) dry chl a in n-hexane (c = 3 x IO-’ M, 1 = 1.Ocm), excitation at 428 mn, (B) wet chl a in n-hexane solution, excitation at 448 mn (1) and at 428 mn (2), and (C) wet chl a in thin films on a quartz slide, excitation at 448 mn.
6
B. Zelent et al./J. Mol. Struct.. 297 (1993) 1-11
absorption spectrum with the maximum between 740 and 745 nm, belonging to the hydrated oligomer. This phenomenon is shown in Fig. 2(A) by comparison of the absorption spectra of dry and wet chl a solutions. The electronic absorption spectrum of the dry solution shows two bands at 428 and 660 nm similar to the absorption spectra of chl a in non-polar solvents [19]. Wet chl a is characterized by the absorption spectrum shifting to lower energy with the maxima at 448 and 745 nm. It is worth noting that the intensity ratio of the blue to red absorption bands is 1.3 for the dry sample and 0.6 for the wet one. The differences in the absorption spectra of dry and wet solutions suggest that chl a in wet n-hexane exists in well-organized aggre-
gate forms. Similar absorption spectra for the pigment in 2,2,4-trimethylpentane and for the electrodeposited wet and dry chl a have also been reported [20,21]. Singlet electronic excitation of dry chl a in dilute solution &,,, = 428 nm) leads to fluorescence emission with a strong band at 665 nm and a low intensity band at 722nm (Fig. 3(A)). Moreover, the fluorescence excitation spectrum (Fig. 4(A)) measured at maximum emission corresponds well to the Soret absorption band of dry chl a in nhexane. This indicates that chl a in dry diluted solution exists in a monomeric form [ 121. The fluorescence emission spectrum of wet chl a solution when excitation is at 448 nm (Fig. 3(B))
‘.
‘--_________------
I
WAVELENGTH
( nm
I
5 0
425
)
WAVELENGTH
I
I
350
425 WAVELENGTH
( nm
)
I 500 ( nm
)
Fig. 4. Fluorescence excitation spectra of (A) dry chl a in n-hexane (c = 3 x lo-’ M, 1 = 1.0 cm), emission at 665 mn, (B) wet chl a in n-hexane solution, emission at 755 nm (1) and 666 nm (2), and (C) wet chl a in thin films on a quartz slide, emission at 756nm.
7
B. Zelent et al./J. Mol. Struct., 297 (1993) 1-11
The electronic absorption spectrum of a thin film of wet chl a (Fig. 2(B)) shows a Soret band at 448nm and a low energy band at 746mn, similar to the spectrum of wet chl a in n-hexane and in LB films [7]. Nevertheless, the emission spectrum of the thin film, when excitation is at 448 nm, exhibits only one band at 756nm (Fig. 3(C)). Moreover, the fluorescence excitation spectrum, when emission is at 756nm (Fig. 4(C)) corresponds to the absorption spectrum of the hydrated chl a thin film. These spectra indicate highly unified hydrated aggregates. The observed lifetime of the emission at 756 nm, 72 = 0.12 f 0.03 ns, accounts for 97% (Table 1). This is also consistent with the presented properties of wet chl a in thin film. The fluorescence lifetime components of wet chl a in monolayers and multilayer LB films, previously reported in ref. 7, correspond well to the lifetime values of wet chl a in thin film (see Table 1). This clearly indicates that the aggregation states of the described molecules are of similar types to those of the model systems. The low energy emission transition has also been measured for multilayers of dry chl a in a watersaturated nitrogen atmosphere [13]. It was observed that the fluorescence maximum of chl a
exhibits two emission bands at 666 and 755nm. However, when excitation is set at 428nm, the fluorescence emission spectrum shows one strong band at 666 nm and two low intensity bands at 720 and 755 nm. These emission spectra, along with the fluorescence excitation spectra of wet chl a in nhexane when emission is at 755 or 666nm (Fig. 4(B)), indicate that the pigment exists not only as a hydrated oligomer but also as hydrated monomers and dimers. Such a conclusion can also be supported by the fluorescence lifetime values presented in Table 1 and Fig. 5. The decay of the emission at 755 nm is biexponential with the lifetime value of q = 5.54 f 0.05ns (30%) and r2 = 0.17 f 0.06 ns (70%). The short lifetime component belongs to well-aggregated forms of chl a [22]. Monoexponential decay has been measured for the 666nm emission band with 71 = 5.68 f 0.02ns. For diluted dry chl a solution, the monoexponential decay of the emission at 665 nm yields r1 = 5.75 f 0.02ns (Table 1); this lifetime value corresponds to emission of the chl a monomer [12]. The difference between dry (pi = 5.75f 0.02 ns) and wet (q = 5.68 f 0.02ns) chl a in n-hexane seems to be due to chl u--water interactions.
Table 1 Fluorescence excitation and emission maxima and fluorescence lifetimes of dry and wet chl a in n-hexane solutions and wet chl a in monolayers, multilayers and thin films on quartz slides Pigment conditions
Excitation
Emission
(nm)
(nm)
[AS,
&,
Dry chl a
428
665
5.75 f 0.02
_
428
666
5.68 f 0.02
_
448
755
(100%) 5.54 f 0.05 (30%)
0.17 f 0.06 (70%)
Wet chl a monolayera
448
755
3.31 f 0.43 (8%)
0.12 f 0.05 (92%)
Wet chl a multilayera
448
755
3.25 f 0.33 (5%)
0.15 f 0.05 (95%)
Wet chl a thin film
448
756
3.22 f 0.74 (3%)
0.12 f 0.03 (97%)
in n-hexane Wet chl a
(100%)
in n-hexane
a From ref. 7.
B.Zelentetal./J. Mol. Struct..297(1993) I-II
z 2
5; CHANNEL
NUMBER
2
CHANNEL
NUMBER
21 = 5.54kO.05 ns
-/
72 = 0.17* 0.06ns
L i 10 TIME
/
NANOSECONDS
Fig. 5. Time-resolved fluorescence exponential decay (X, = 666mn)
TIME
/
20 30 NANOSECONDS
40
decay curves (...) associated with the lamp profiles (- - - - -) showing (A) the single and (B) the biexponential decay (X, = 755 nm) of wet chl a in n-hexane. Excitation wavelength, 440 f 50 nm.
in a wet atmosphere gradually shifts from 740 to 760mn when the temperature decreases from 298 to 85 K. Moreover, Kooyman et al. [6] have studied chl u-water complexes in n-octane, using fluorescence detected magnetic resonance and fluorimetry. From the concentration dependence of the pigment fluorescence at 4.2K, they detected four emission bands at 669,687,725 and 750 nm. These bands have been assigned to the emission of (chl a - HzO), (chl a - 2H20), (chl a - H20)2 and (chl a - H20), species correspondingly. It is well known that chl a molecules form selfaggregates via the ring V Cs keto C =O.. .Mg interactions and chl u-nucleophile ligand adducts via electron donor-acceptor interactions [3,4]. In aliphatic or cycloaliphatic hydrocarbon solvents, chl a can form chl u-chl a aggregates even in the most diluted solutions. However, in the presence of
water, which is a polar molecule, the dimers or oligomers of chl a can be easily disrupted to form hydrated monomers and oligomers like (chl a. H,O), or (chl a - 2H20),. FT-IR spectroscopy has been used to study the different aggregation states of chl a [3,23-261. The band at 1733-l 735 cm-’ is the vibration frequency of the C=O ester group positioned at Cl0 in the porphyrin ring. Another important band in that region is situated at 1688-1692 cm-‘. It corresponds to the vibration of the free (uncoordinated) Cg keto C = 0 group. The 1659-1661 cm-’ region is related to the frequency of the coordinated Cg keto C = 0 group, which is bound to the magnesium of another chl a molecule by the oxygen of the carbonyl functional group in dry dimers or oligomers. The fourth important band in the C=O vibration region is the one appearing at
B. Zelent et al./J. Mol. Struct., 297 (1993) l-11
I
;
.
3
,._. -*_
,*-k.,.’ f’ .J.
i
i i ‘\ \ ‘\
‘Y
I
600
700 WAVELENGTH
(nm )
800
I I I 1700 1600 1800 WAVE NUMBER ( cm-9
Fig. 6. Electronic absorption (A) and FT-IR spectra (B) of thin films of hydrated chl a oligomers: on cleaned CaFz crystal (1); after 18 h vacuum on cleaned CaF2 (2); on Cd arachidate covered CaF2 (3).
B. Zelent et al/J. Mol. Strut., 297 (1993) 1-11
10
1633-1647 cm-‘. Its interpretation has been subject to discussion. It may be the frequency range of the carbonyl functional group in the Cs keto C = 0. . -H-O(H). . .Mg hydrated aggregation state, which is observed in hydrated oligomers [27], or it may be the bending mode of the water molecule that is trapped between the two chl a molecules in the same aggregation state as indicated before [3]. In the case of amorphous thin films of hydrated oligomers, the absorption spectrum which shows a strong maximum at 746 nm (Fig. 6, curve Al) indicates a strong oligomer concentration. The first IR spectrum (curve Bl) supports the presence of a strong water band at 1639 cm-‘, completely overlapping the aggregated Cs keto C = 0. . .Mg band (which may be observed around 1660 cm-’ in less concentrated samples in hydrated oligomers [7]). Drying the sample for 18 h under a strong vacuum (curves A2 and B2) produced a small decrease in the water band in the IR spectrum due to the removal of excess water that was trapped in the sample during its preparation. However, the water band is still very strong, indicating that the water molecules present in the sample are strongly coordinated to the chls in the hydrated oligomeric form. The last IR and absorption spectra (curves A3 and B3) were produced from the same wet oligomer solution as the other amorphous samples, 24 h after the two previous spectra. These spectra show that the sample was more concentrated in the hydrated oligomers. Since oligomers are strongly adsorbed onto glass surfaces, a continual decrease in the concentration of oligomers in solution is usually observed with time as they are adsorbed on the walls of the vials in which the solution is stored. We were at first very surprised that our FT-IR results showed otherwise. One way to explain this behavior is that the CaF2 crystals somehow destroy our hydrated samples. It may be possible that the Cd arachidate layers that covered the crystal in that last experiment protected our oligomer sample from destruction. This protection effect was not observed in LB multilayer films of wet chl a oligo-
mers deposited on layers of Cd arachidate [7]. We believe that as oligomers are attracted to glass surfaces, they could be repulsed by Cd arachidate covered CaF2 crystals when an LB technique is used to deposit oligomers onto the crystal surface. Published results [7] show that wet oligomers are more easily deposited directly on the CaFz hydrophilic surface compared to the hydrophobic Cd arachidate covered crystal. This is possible since it was observed that oligomers are attracted to hydrophilic surfaces. However, the dry chl u molecules somehow have an advantage over the oligomers when the cleaned or Cd arachidate covered CaF2 crystal is dipped into the subphase during LB depositions. This effect could be coupled to a difftcult conservation of the hydrated oligomers on the interface. Using a different deposition technique for the sample on the CaF2 crystal, our present results show that the ratio of wet oligomer to dry chl a is greatly enhanced. In fact, Cd arachidate layers which decreased the LB deposition ratio between the hydrated oligomer and dry chl a now protects the sample from a destructive contact between oligomers and the CaF2 crystal surface. Conclusions
The interfacial tension of dry and wet chl a at the octane-water interface indicates that the properties of this pigment in dry non-polar organic solvents differ from those of wet non-polar organic solvents. This is probably due to the state of chl a molecules which can exist as non-hydrated or hydrated monomers, dimers and/or oligomers. The dry and wet chl a molecules are surface active compounds with different adsorption properties. It can also be seen from interfacial tension measurements that the adsorbed molecules of dry chl a have attraction interaction and wet chl a oligomers show repulsion between clusters. From photophysical studies such as absorption, fluorescence and FT-IR spectroscopies of thin films and from circular dichroism [28] and electron paramagnetic resonance [29] studies, we conclude that wet chl a forms self-organized molecular assemblies
B. Zelent et al./J. Mol. Struct.. 297 (1993) 1-11
consisting of six chl a molecules and some bonded water molecules. Such organized aggregated forms of the wet chl a molecules are characterized by low energy electronic transitions with an absorption band between 740 and 745 nm and a fluorescence band at around 755nm. The lifetime of the emission is 0.1 to 0.2 ns. Acknowledgments This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Fonds pour la Formation de Chercheurs et 1’Aide a la Recherche (FCAR). Financial support as postgraduate scholarships came from FCAR and from La Fondation CEU (UQTR) to J.G. References K.D. Philipson, SC. Tsai and K. Sauer, J. Phys Chem., 75 (1971) 1440. C. Weiss, J. Mol. Spectrosc., 44 (1972) 37. K. Ballschmiter and J.J. Katz, J. Am. Chem. Sot., 91 (1969) 2661. K. Balls&miter and J.J. Katz, Biochim. Biophys. Acta, 256 (1972) 307. A. Agostiano, P. Cosma and M. Della Monica, J. Photochem. Photobiol., A: Chem., 58 (1991) 201. R.P.H. Kooyman, T.J. Schaafsma and J.F. Kleibeuker, Photochem. Photobiol., 26 (1977) 235. G. Munger, R.M. Leblanc, B. Zelent, A.G. Volkov, M.I. Gugeshashvili, J. Gallant, H.-A. Tajmir-Riahi and J. Aghion, Thin Solid Films, 210/211 (1993) 739. 8 F. Lamarche, G. Picard, F. Techy, J. Aghion and R.M. Leblanc, Eur. J. Biochem., 197 (1991) 529. 9 A.G. Volkov, V.D. Kolev, A.L. Levin and L.I. Boguslavsky, Photobiochem. Photobiophys., 10 (1985) 105. 10 J.J. Katz, L.L. Shipman, T.M. Cotton and T.R. Janson, in D. Dolphin (Ed.), The Porphyrins, Vol. 5, Academic Press, New York, 1978, pp. 401458.
11 11 F.K. Fong, in F.K. Fong (Ed.), Light Reaction Path of Photosynthesis, Springer-Verlag, Berlin, 1982, pp. 277-321. 12 J.S. Connolly, A.F. Janzen and E.B. Samuel, Photothem. Photobiol., 36 (1982) 559. 13 S. Krawczyk, R.M. Leblanc and L. Marcotte, J. Chim. Phys., 85 (1988) 1073. 14 J.J. Katz, J.R. Norris, L.L. Shipman, M.C. Thurnauer and M.R. Wasielewski, Annu. Rev. Biophys. Bioeng., 7 (1978) 393. 15 M.R. Wasielewski, in F.K. Fong (Ed.), Light Reaction Path of Photosynthesis, Springer-Verlag, Berlin, 1982, pp. 234276. 16 L.I. Bognslavsky and A.G. Volkov, in V.E. Kazarinov (Ed.), The Interface Structure and Electrochemical Processes at the Boundary Between Two Immiscible Liquids, Springer-Verlag, Berlin, 1987, pp. 143-178. 17 R.M. Leblanc, P.-F. Blanchet, D. Cot&, M.I. Gugeshashvili, G. Munger and A.G. Volkov, in SPIE-Int. Sot. Opt. Eng. (Ed.), Photochemistry and Photoelectrochemistry of Organic and Inorganic Molecular Thin Films, Vol. 1436, Bellingham, WA, 1991, pp. 92-102. 18 G. Munger, L. Lorrain, G. Gagne and R.M. Leblanc, Rev. Sci. Instrum., 58 (1987) 285. 19 L. Szalay, G.S. Singhal, E. Tombacz and L. Kozma, Acta Phys. Acad. Sci. Hung., 34 (1973) 341. 20 C.W. Tang and A.C. Albrecht, Mol. Cryst. Liq. Cryst., 25 (1974) 53. 21 J.P. Dodelet, J. Le Brech and R.M. Leblanc, Photothem. Photobiol., 29 (1979) 1135. 22 A.J. Alfano, F.E. Lytle, M.S. Showell and F.K. Fong, J. Chem. Phys., 82 (1985) 758. 23 T.M. Cotton, P.A. Loach, J.J. Katz and K. Ballschmitter, Photochem. Photobiol., 27 (1978) 735. 24 C. Chapados, D. Get-main and R.M. Leblanc. Biophys. Chem., 12 (1980) 189. 25 C. Chapados and R.M. Leblanc, Chem. Phys. Lett., 49 (1977) 180. 26 G.R. Seely, in J. Barber (Ed.), Primary Processes of Photosynthesis, Elsevier, New York, 1977, pp. l-53. 27 J.P. Dodelet, J. Le Brech, C. Chapados and R.M. Leblanc, Photochem. Photobiol., 31 (1980) 143. 28 M.D. Kandelaki, A.G. Volkov, V.V. Shubin and L.I. Boguslavsky, Biochim. Biophys. Acta, 893 (1987) 170. 29 A.G. Volkov, M.I. Gugeshashvili, G. Munger and R.M. Leblanc, Bioelectrochem. Bioenerg., 29 (1993) 305.
Hydrated chlorophyll a oligomers in solutions, monolayers, and thin films Bogumil Zelent, Judith Gallant, Alexander G. Volkov’, Maya I. Gugeshashvili2, Gaktan Munger, Heidar-Ali Tajmir-Riahi, Roger M. Leblanc* Centre de recherche en photobiophysique, Universite’ du Qukbec h Trois-Rivi&es, 3351, boul. des Forges, C.P. 500, Trois-Rivieres, Que. G9A 5H7, Canada (Received 2 November 1992) Abstract The interfacial tension measurements of dry and wet chlorophyll a at an octane-water interface show differences in adsorption behavior of the pigment depending on its aggregation state. Optical properties were also investigated using absorption and Fourier transform-infrared spectroscopy, along with fluorescence (excitation and emission) measurements in non-polar solvents and in thin films. The absorption and fluorescence spectral characteristics, and fluorescence lifetime measurements of dry and wet samples clearly indicate the difference in aggregation properties of this photosynthetic pigment. A strong decrease in the energy of QY electronic transitions (Av = 1700cm-‘), due to the formation of hydrated oligomers, was accompanied by a decrease in fluorescence lifetime from 5.75 f 0.02ns (diluted dry chlorophyll a in n-hexane) to 0.12 f 0.03 ns (wet chlorophyll a in thin films). Discussion of these results is based on the key role of the central magnesium atom in the porphyrin ring in intermolecular interactions and self-organization of chlorophyll a molecules.
Introduction The electronic charge distribution of the r-electron conjugated system in the porphyrin nuclear framework of chlorophyll a (chl a) is highly sensitive to environmental interactions. This is manifested by the energy levels and the probability of electronic transitions from the ground to the singlet excited electronic states which are polarized along the x and y axes of the porphyrin ring [1,2]. The changes in the electronic absorption spectra of chl a caused by intermolecular interactions like chl a-chl a or chl a-water [3-71 and chl a-protein [8] in non*Corresponding author. Tel: (819) 376-5077.Fax: (819) 376-5057.E-mail:[email protected] CA. ’ Dedicated to Professor CamilleSandorfy. ‘Permanent address: The A.N. Frumkin Institute of Electrochemistry,Acad. Sci. USSR, 31 Leninsky Prospekt, Moscow 117071,Russian Federation.
polar and polar solvents have been widely studied in view of the numerous roles played by chl a in the primary events of plant photosynthesis: light harvesting, energy transfer and charge separation in photosynthetic units [g-17]. The state of chl a molecules in green leaves is believed to be in different aggregated forms and is still under investigation. The interface between two immiscible liquids with immobilized photosynthetic pigments can serve as the simplest model of a biological membrane convenient for the investigation of photoprocesses that are accompanied by spatial separation of charges. It was demonstrated by mass spectroscopy and polarographic studies [16] that on illumination of wet chl a at an octane-water interface, oxygen evolution with a quantum efficiency of lo20% takes place in the presence of proton acceptors (pentachlorophenol or 2,4-dinitrophenol) in
2
octane and electron acceptors (nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide phosphate (NADP+) or K3[Fe(CN)6]) in water. This process is accompanied by the appearance of a photopotential at the octane-water interface. The process occurs at the interface and the photopotential and reaction rate are proportional to the surface concentration of chl a. However, optically transparent electrodes, modified with monolayers of photosynthetic pigments using the Langmuir-Blodgett (LB) technique, are also suitable for the investigation of chemical models of photosynthesis [17]. During photocatalytic oxidation of hydroquinone by chl a or pheophytin a in monolayers, the standard Gibbs free energy of electrochemical reaction is negative and hence solar energy was used to overcome the activation barrier. In the absence of hydroquinone, it is possible to use water molecules as electron donors and NADP+ or methyl viologen as an electron acceptor. In this case solar energy can be used to perform work, since the standard Gibbs free energy of the reaction of water photooxidation and NADP+ reduction is positive. This process of artificial photosynthesis is the chemical model of natural photosynthesis. The utilization of solar energy for the transport of electrons from water to NADP+ is the most important process in the bioenergetics of natural photosynthesis. Considering the importance of chemical models of photosynthesis, in this paper we have investigated the interfacial tension at the octane-water interface as well as the optical properties of dry and wet chl a. Some new spectroscopic results for dry and wet samples in solutions and in thin films are presented and discussed with regard to their different structural organization.
Experimental Samples and reagents
Chl a was purchased from Fluka Chemical Corp. (Ronkonkoma, NY) in a 1 mg ampoule, and
B. Zelent et al.lJ. Mol. Struct., 297 (1993) l-11
arachidic acid from Applied Science Laboratories Inc. (State College, PA). The deionized water (Nanopure filter system, Barnsted, Boston, MA) used to prepare all the solutions was doubly distilled in quartz (Model Bi 18, HeraeusQuarzschmelze, Hanau, Germany). Its specific resistivity was 18 MRcm, and its surface tension was higher than 71 mN m-l. Benzene from Anachemia Accusolv (Montreal, Que.), used to produce dry chl a solutions, was distilled at 80°C in a 3Ocm Vigreux column. nHexane was purchased from Aldrich (Milwaukee, WI), and chloroform from Anachemia Accusolv (Montreal, Que.). They were both distilled before use. n-Octane was bought from BDH Chemicals Canada Ltd. (Toronto, Ont.) and Fluka Chemie AG (Buchs, Switzerland). The Fourier transform-infrared (FT-IR) spectra of the hydrated chl a oligomers were measured in thin films. The substrate was a 35 x 9 x 2mm CaF2 crystal. Its cleaning procedure included soaking in a phosphate free, non-ionic, neutral pH liquid Aquet detergent, purchased from Manostat Laboratory Instruments and Scientific Apparatus (New York), followed by rinsing in chloroform and in methanol. All these steps were carried out in an ultrasonic bath which was regularly filled with ice to prevent overheating since CaF2 is sensitive to thermal shock. All experiments were performed at a temperature of 20°C under dim green light.
Hydrated chlorophyll a oligomer preparation
Wet n-hexane (or n-octane) was prepared with equal volumes of the solvent and water equilibrated for 48 h. After equilibration the solvent was transferred to a vial containing chl a and was sonicated for 100min in a cooled ultrasonic bath. During sonication, the formation of hydrated oligomer was controlled by UV/visible spectroscopy. The usual concentration of hydrated chl a solutions was 2 x 10m5M.
3
B. Zelent et al./J. Mol. Struct.. 297 (1993) I-11
Interfacial tension measurements The interfacial tension at the octane-water interface was measured by determining the weight and volume of the drop falling from the end of a capillary tube under its own gravity. The surface tension (y) was obtained from Eq. (1) Y = Vdl
(1)
- d2)Wr
where d, and d2 are densities of the liquid studied, V the drop volume, g the acceleration of gravity, F a correction function found in the Harkins-Broun table, and I the capillary radius. The equipment used to measure the interfacial tension consisted of a glass capillary, a 1 cm3 syringe, a micrometer and a vessel filled by a liquid with lower density used to immerse the capillary. The internal capillary diameter was 0.6 mm. The drop lifetime necessary to establish adsorption equilibrium was not less than 4min.
Langmuir-BlodgettJ
deposition
In the case of some FT-IR measurements, multilayers of cadmium (Cd) arachidate were deposited on the freshly cleaned CaF2 substrate. A total of ten monolayers were deposited (five on each side). The Langmuir trough was described elsewhere [ 181. The subphase used to spread arachidic acid was a 10e3 M tris(hydroxymethyl)aminomethane Trizma base buffer set at pH 6.5, with 10T4M cadmium chloride. Spreading solvent was freshly distilled degassed chloroform. Room and subphase temperatures were maintained at 20°C during deposition. The surface pressure for LB deposition was set at 30mNm-‘.
Preparation of thin jilms Thin films of hydrated chl a oligomers were prepared for electronic absorption and fluorescence spectroscopies. The quartz slides were dipped in the solution of hydrated oligomers which were
easily adsorbed onto the surface and dried with a slow stream of argon. Thin films of hydrated oligomers were also investigated by FT-IR spectroscopy. They were prepared by a solvent evaporation technique. The hydrated oligomer solutions were directly deposited on the surface of a Cd arachidate covered or a freshly cleaned CaF2 crystal. Each drop was dried with a flow of argon or nitrogen before adding some new material to the surface to prevent its oxidation.
Photophysical measurements Absorption spectra were performed from 350 to 800nm with a Hewlett-Packard diode array spectrophotometer, model 8452A (Palo Alto, CA), controlled by an IBM personal system/2 computer. In the case of hydrated oligomer thin films deposited on CaF2 (those used for FT-IR spectroscopy), the absorption reference spectra of the Cd arachidate or the cleaned CaF2 substrate were subtracted from the absorption spectra. The fluorescence emission and excitation spectra of dry and wet chl a solutions and thin films on quartz slides were registered by means of a Spex Fluorolog II spectrofluorimeter, model 1870 (Spex Industries Inc., Metuchen, NJ), equipped with a Datamate DM 1 data acquisition system and a thermoelectrically cooled Hamamatsu, model R928 photomultiplier tube (Hamamatsu Photonic Systems Corp., Bridgewater, NJ). The spectra were corrected for the lamp intensity and for the photomultiplier sensitivity. The fluorescence lifetimes of the same samples were measured using Photochemical Research Associates International Inc. Model 3000 fluorescence lifetime, single photon counting equipment (PRA, London, Ont.). The excitation light was generated by nitrogen flash lamp PRA model 510. The samples were illuminated in the spectral region of 440 f 50 nm using a bandpass filter (Oriel, Stratford, CT). Fluorescence emission was detected at 670 f 5 nm or 750 f 5 nm using bandpass interfer-
B. Zelent et al./J. Mol. Strut.,
0
’
I
1
100
200
I
AREA
Fig. 1. The dependence
of surface pressure at the octane-water
ence filters (Ditric Optics Inc., Hudson, MA). Data were analyzed by PRA statistical deconvolution program. The infrared (IR) spectra were recorded on a Bomem DA3.02 FT-IR spectrophotometer (Vanier, Que.), equipped with a liquid nitrogen cooled HgCdTe detector. A total of 500 scans was carried out with a resolution of 4cm-‘. Baseline correction and smoothing procedure were also
I
300
297 (1993)
I-11
I
400
500
(A*)
interface on surface concentration
of dry (1) and wet chl a (2).
performed. The reference spectra were recorded with Cd arachidate covered or with cleaned CaFz crystals depending on whether or not the sample contained the Cd arachidate layers. Results and discussion Chl a molecules are known to have an abundant surface activity and therefore, they considerably
LOS-
7457::
A
I i: ,
0.64.
400
500 600 WAVELENGTH
Fig. 2. Electronic absorption
700 (nm)
6f
400
500 600 WAVELENGTH,
700
601
spectra of (A) wet (1) and dry chl a (2) in n-hexane solution and (B) wet chl a in thin films on a quartz slide.
5
B. Zelent et al./J. Mol. Struct., 297 (1993) 1-11
reduce the tension during adsorption at the interface This property was used to determine the surface excess of chl a according to Gibbs theory. Figure 1 shows the dependence of interfacial tension on the chl a concentration in dry (curve 1) and wet (curve 2) n-octane. A gradual reduction of y can be observed in the chl a concentration ranges from 1 x 10e6 to 5 x 10m6M for wet and from 1 x low6 to 1.5 x lo-’ M for dry solutions. For the dry solution, the standard free Gibbs energy of adsorption equilibrium is equal to -27.4 kJ mol-’ . For hydrated oligomers, the standard free Gibbs energy is equal to -35.2 kJ mol-‘. The positive attraction constant a = 0.59 for dry chl a shows the attraction interaction between the adsorbed molecules. The negative constant
a = -0.215 indicated repulsion between clusters of hydrated oligomers. The difference between the two curves is in the hydration state of the chl a solutions and the structural organization of the adsorbed particles. This is a rare example in colloid chemistry where a surface active compound can be found in two different adsorbed states, as dry monomeric molecules and as self-organized hydrated molecular clusters. The investigations on biologically active compounds from membranes of chloroplasts, mitochondria and bacteria led us to study a new type of surface active compound, capable of forming self-organized molecular assemblies. When chl a is dissolved in the water-saturated hydrocarbon, an additional band appears in the
A
I
WAVELENGTH (nm)
WAVELENGTH ( nm )
WAVELENGTH
(nm 1
Fig. 3. Corrected fluorescence spectra of (A) dry chl a in n-hexane (c = 3 x IO-’ M, 1 = 1.Ocm), excitation at 428 mn, (B) wet chl a in n-hexane solution, excitation at 448 mn (1) and at 428 mn (2), and (C) wet chl a in thin films on a quartz slide, excitation at 448 mn.
6
B. Zelent et al./J. Mol. Struct.. 297 (1993) 1-11
absorption spectrum with the maximum between 740 and 745 nm, belonging to the hydrated oligomer. This phenomenon is shown in Fig. 2(A) by comparison of the absorption spectra of dry and wet chl a solutions. The electronic absorption spectrum of the dry solution shows two bands at 428 and 660 nm similar to the absorption spectra of chl a in non-polar solvents [19]. Wet chl a is characterized by the absorption spectrum shifting to lower energy with the maxima at 448 and 745 nm. It is worth noting that the intensity ratio of the blue to red absorption bands is 1.3 for the dry sample and 0.6 for the wet one. The differences in the absorption spectra of dry and wet solutions suggest that chl a in wet n-hexane exists in well-organized aggre-
gate forms. Similar absorption spectra for the pigment in 2,2,4-trimethylpentane and for the electrodeposited wet and dry chl a have also been reported [20,21]. Singlet electronic excitation of dry chl a in dilute solution &,,, = 428 nm) leads to fluorescence emission with a strong band at 665 nm and a low intensity band at 722nm (Fig. 3(A)). Moreover, the fluorescence excitation spectrum (Fig. 4(A)) measured at maximum emission corresponds well to the Soret absorption band of dry chl a in nhexane. This indicates that chl a in dry diluted solution exists in a monomeric form [ 121. The fluorescence emission spectrum of wet chl a solution when excitation is at 448 nm (Fig. 3(B))
‘.
‘--_________------
I
WAVELENGTH
( nm
I
5 0
425
)
WAVELENGTH
I
I
350
425 WAVELENGTH
( nm
)
I 500 ( nm
)
Fig. 4. Fluorescence excitation spectra of (A) dry chl a in n-hexane (c = 3 x lo-’ M, 1 = 1.0 cm), emission at 665 mn, (B) wet chl a in n-hexane solution, emission at 755 nm (1) and 666 nm (2), and (C) wet chl a in thin films on a quartz slide, emission at 756nm.
7
B. Zelent et al./J. Mol. Struct., 297 (1993) 1-11
The electronic absorption spectrum of a thin film of wet chl a (Fig. 2(B)) shows a Soret band at 448nm and a low energy band at 746mn, similar to the spectrum of wet chl a in n-hexane and in LB films [7]. Nevertheless, the emission spectrum of the thin film, when excitation is at 448 nm, exhibits only one band at 756nm (Fig. 3(C)). Moreover, the fluorescence excitation spectrum, when emission is at 756nm (Fig. 4(C)) corresponds to the absorption spectrum of the hydrated chl a thin film. These spectra indicate highly unified hydrated aggregates. The observed lifetime of the emission at 756 nm, 72 = 0.12 f 0.03 ns, accounts for 97% (Table 1). This is also consistent with the presented properties of wet chl a in thin film. The fluorescence lifetime components of wet chl a in monolayers and multilayer LB films, previously reported in ref. 7, correspond well to the lifetime values of wet chl a in thin film (see Table 1). This clearly indicates that the aggregation states of the described molecules are of similar types to those of the model systems. The low energy emission transition has also been measured for multilayers of dry chl a in a watersaturated nitrogen atmosphere [13]. It was observed that the fluorescence maximum of chl a
exhibits two emission bands at 666 and 755nm. However, when excitation is set at 428nm, the fluorescence emission spectrum shows one strong band at 666 nm and two low intensity bands at 720 and 755 nm. These emission spectra, along with the fluorescence excitation spectra of wet chl a in nhexane when emission is at 755 or 666nm (Fig. 4(B)), indicate that the pigment exists not only as a hydrated oligomer but also as hydrated monomers and dimers. Such a conclusion can also be supported by the fluorescence lifetime values presented in Table 1 and Fig. 5. The decay of the emission at 755 nm is biexponential with the lifetime value of q = 5.54 f 0.05ns (30%) and r2 = 0.17 f 0.06 ns (70%). The short lifetime component belongs to well-aggregated forms of chl a [22]. Monoexponential decay has been measured for the 666nm emission band with 71 = 5.68 f 0.02ns. For diluted dry chl a solution, the monoexponential decay of the emission at 665 nm yields r1 = 5.75 f 0.02ns (Table 1); this lifetime value corresponds to emission of the chl a monomer [12]. The difference between dry (pi = 5.75f 0.02 ns) and wet (q = 5.68 f 0.02ns) chl a in n-hexane seems to be due to chl u--water interactions.
Table 1 Fluorescence excitation and emission maxima and fluorescence lifetimes of dry and wet chl a in n-hexane solutions and wet chl a in monolayers, multilayers and thin films on quartz slides Pigment conditions
Excitation
Emission
(nm)
(nm)
[AS,
&,
Dry chl a
428
665
5.75 f 0.02
_
428
666
5.68 f 0.02
_
448
755
(100%) 5.54 f 0.05 (30%)
0.17 f 0.06 (70%)
Wet chl a monolayera
448
755
3.31 f 0.43 (8%)
0.12 f 0.05 (92%)
Wet chl a multilayera
448
755
3.25 f 0.33 (5%)
0.15 f 0.05 (95%)
Wet chl a thin film
448
756
3.22 f 0.74 (3%)
0.12 f 0.03 (97%)
in n-hexane Wet chl a
(100%)
in n-hexane
a From ref. 7.
B.Zelentetal./J. Mol. Struct..297(1993) I-II
z 2
5; CHANNEL
NUMBER
2
CHANNEL
NUMBER
21 = 5.54kO.05 ns
-/
72 = 0.17* 0.06ns
L i 10 TIME
/
NANOSECONDS
Fig. 5. Time-resolved fluorescence exponential decay (X, = 666mn)
TIME
/
20 30 NANOSECONDS
40
decay curves (...) associated with the lamp profiles (- - - - -) showing (A) the single and (B) the biexponential decay (X, = 755 nm) of wet chl a in n-hexane. Excitation wavelength, 440 f 50 nm.
in a wet atmosphere gradually shifts from 740 to 760mn when the temperature decreases from 298 to 85 K. Moreover, Kooyman et al. [6] have studied chl u-water complexes in n-octane, using fluorescence detected magnetic resonance and fluorimetry. From the concentration dependence of the pigment fluorescence at 4.2K, they detected four emission bands at 669,687,725 and 750 nm. These bands have been assigned to the emission of (chl a - HzO), (chl a - 2H20), (chl a - H20)2 and (chl a - H20), species correspondingly. It is well known that chl a molecules form selfaggregates via the ring V Cs keto C =O.. .Mg interactions and chl u-nucleophile ligand adducts via electron donor-acceptor interactions [3,4]. In aliphatic or cycloaliphatic hydrocarbon solvents, chl a can form chl u-chl a aggregates even in the most diluted solutions. However, in the presence of
water, which is a polar molecule, the dimers or oligomers of chl a can be easily disrupted to form hydrated monomers and oligomers like (chl a. H,O), or (chl a - 2H20),. FT-IR spectroscopy has been used to study the different aggregation states of chl a [3,23-261. The band at 1733-l 735 cm-’ is the vibration frequency of the C=O ester group positioned at Cl0 in the porphyrin ring. Another important band in that region is situated at 1688-1692 cm-‘. It corresponds to the vibration of the free (uncoordinated) Cg keto C = 0 group. The 1659-1661 cm-’ region is related to the frequency of the coordinated Cg keto C = 0 group, which is bound to the magnesium of another chl a molecule by the oxygen of the carbonyl functional group in dry dimers or oligomers. The fourth important band in the C=O vibration region is the one appearing at
B. Zelent et al./J. Mol. Struct., 297 (1993) l-11
I
;
.
3
,._. -*_
,*-k.,.’ f’ .J.
i
i i ‘\ \ ‘\
‘Y
I
600
700 WAVELENGTH
(nm )
800
I I I 1700 1600 1800 WAVE NUMBER ( cm-9
Fig. 6. Electronic absorption (A) and FT-IR spectra (B) of thin films of hydrated chl a oligomers: on cleaned CaFz crystal (1); after 18 h vacuum on cleaned CaF2 (2); on Cd arachidate covered CaF2 (3).
B. Zelent et al/J. Mol. Strut., 297 (1993) 1-11
10
1633-1647 cm-‘. Its interpretation has been subject to discussion. It may be the frequency range of the carbonyl functional group in the Cs keto C = 0. . -H-O(H). . .Mg hydrated aggregation state, which is observed in hydrated oligomers [27], or it may be the bending mode of the water molecule that is trapped between the two chl a molecules in the same aggregation state as indicated before [3]. In the case of amorphous thin films of hydrated oligomers, the absorption spectrum which shows a strong maximum at 746 nm (Fig. 6, curve Al) indicates a strong oligomer concentration. The first IR spectrum (curve Bl) supports the presence of a strong water band at 1639 cm-‘, completely overlapping the aggregated Cs keto C = 0. . .Mg band (which may be observed around 1660 cm-’ in less concentrated samples in hydrated oligomers [7]). Drying the sample for 18 h under a strong vacuum (curves A2 and B2) produced a small decrease in the water band in the IR spectrum due to the removal of excess water that was trapped in the sample during its preparation. However, the water band is still very strong, indicating that the water molecules present in the sample are strongly coordinated to the chls in the hydrated oligomeric form. The last IR and absorption spectra (curves A3 and B3) were produced from the same wet oligomer solution as the other amorphous samples, 24 h after the two previous spectra. These spectra show that the sample was more concentrated in the hydrated oligomers. Since oligomers are strongly adsorbed onto glass surfaces, a continual decrease in the concentration of oligomers in solution is usually observed with time as they are adsorbed on the walls of the vials in which the solution is stored. We were at first very surprised that our FT-IR results showed otherwise. One way to explain this behavior is that the CaF2 crystals somehow destroy our hydrated samples. It may be possible that the Cd arachidate layers that covered the crystal in that last experiment protected our oligomer sample from destruction. This protection effect was not observed in LB multilayer films of wet chl a oligo-
mers deposited on layers of Cd arachidate [7]. We believe that as oligomers are attracted to glass surfaces, they could be repulsed by Cd arachidate covered CaF2 crystals when an LB technique is used to deposit oligomers onto the crystal surface. Published results [7] show that wet oligomers are more easily deposited directly on the CaFz hydrophilic surface compared to the hydrophobic Cd arachidate covered crystal. This is possible since it was observed that oligomers are attracted to hydrophilic surfaces. However, the dry chl u molecules somehow have an advantage over the oligomers when the cleaned or Cd arachidate covered CaF2 crystal is dipped into the subphase during LB depositions. This effect could be coupled to a difftcult conservation of the hydrated oligomers on the interface. Using a different deposition technique for the sample on the CaF2 crystal, our present results show that the ratio of wet oligomer to dry chl a is greatly enhanced. In fact, Cd arachidate layers which decreased the LB deposition ratio between the hydrated oligomer and dry chl a now protects the sample from a destructive contact between oligomers and the CaF2 crystal surface. Conclusions
The interfacial tension of dry and wet chl a at the octane-water interface indicates that the properties of this pigment in dry non-polar organic solvents differ from those of wet non-polar organic solvents. This is probably due to the state of chl a molecules which can exist as non-hydrated or hydrated monomers, dimers and/or oligomers. The dry and wet chl a molecules are surface active compounds with different adsorption properties. It can also be seen from interfacial tension measurements that the adsorbed molecules of dry chl a have attraction interaction and wet chl a oligomers show repulsion between clusters. From photophysical studies such as absorption, fluorescence and FT-IR spectroscopies of thin films and from circular dichroism [28] and electron paramagnetic resonance [29] studies, we conclude that wet chl a forms self-organized molecular assemblies
B. Zelent et al./J. Mol. Struct.. 297 (1993) 1-11
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