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Spectral properties of bacteriochlorophyll c in nematic liquid crystal. Part 1. Monomeric forms of dye
SPECTROCHIMICA ACTA PART A
ELSEVIER
Spectrochimica Acta Part A 52 (1996) 251 264
Spectral properties of bacteriochlorophyll c in nematic liquid crystal. Part 1. Monomeric forms of dye A. D u d k o w i a k a,,, C. F r a n c k e a, J. A m e s z ~, A. Planner b, I. H a n y 2 b, D. F r o c k o w i a k b Department ()[" Biophysics, Huygens Laboratory, University (~["Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands b Moleeular Physies Laboratory, Institute o/' Physics, Poznafi University of Technology Piotrowo 3, 60-965 Poznah, Poland
Received 31 May 1995; accepted in final form 21 September 1995
Abstract
The spectroscopic features of bacteriochlorophyll c and bacteriopheophytin c in a nematic liquid crystal matrix have been investigated. Absorption, circular dichroism, fluorescence and time resolved delayed luminescence spectra have been measured. The pigment is introduced to the liquid crystal from a dry and from a hydrated chloroform solution. In both cases the pigment is in the monomeric form. Hydration of the solvent and the presence or absence of the central Mg atom affect the interaction of the pigment molecules with the liquid crystal matrix, changing the fluorescence anisotropy. A model for the bacteriochlorophyll c orientation in the liquid crystal is proposed and the averaged angles between the transition moments and the liquid crystal orientation axis are determined. A slow process (in the microsecond range) of radiative deactivation of energy absorbed by the pigments is observed. This delayed emission could be due to pigment ionization and delayed charge recombination and/or thermal activation from the triplet to the excited singlet state. Keywords: Bacteriochlorophyll c; Bacteriopheophytin c; Delayed luminescence; Liquid crystal; Polarized spectroscopy
Abbreviations
BChl, b a c t e r i o c h l o r o p h y l l ; [E,E] BChl c v, 8,12-diethyl BChl c esterified with farnesol (F); [Pr,E] BChl Cv, 8 - n - p r o p y l - 1 2 - e t h y l BChl c esterified with farnesol (F); BPhe, b a c t e r i o p h e o p h y t i n ; C D , c i r c u l a r dichroism; 6 C H B T , 4* Corresponding author at Molecular Physics Laboratory, Institute of Physics, Poznafi University of Technology, Piotrowo 3, 60-965 Poznafi, Poland.
(trans-4'-n-hexylcyclohexyl)-isothiocyanato-benz-
ene; Chl, c h l o r o p h y l l ; D L , d e l a y e d luminescence; E B B A , p - e t h o x y b e n z o l i d e n o - p ' - b u t y l a n i l i n e ; F W H M , full w i d t h at h a l f m a x i m u m ; LC, liquid crystal; MBBA, p-methoxy-benzylideno-p'butylaniline; T M , t r a n s i t i o n m o m e n t .
1. I n t r o d u c t i o n
0584-8539/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved S S D I 0584-8539(95)01577-9
B a c t e r i o c h l o r o p h y l l c (BChl
c) is the m a i n
252
A. Dudkowiak et al. / Spectrachimica Acta Part A 52 (1996) 251 264
light-absorbing pigment in various species of green sulfur bacteria [1,2]. It is present in chlorosomes which are attached to the membrane through the F e n n a - M a t t h e w s Olson (FMO) complex [3,4]. BChl c is oriented to a high degree in chlorosomes [5-9]. The spatial arrangement of the pigment molecules, their mutual interactions and their interactions with the chlorosomal proteins have not yet been fully elucidated [9-11]. It thus appeared of interest to study a simpler ordered model simulating some aspects of the properties of the native system. We therefore tried to mimic the natural situation, where the BChls c are oriented in a regular manner [5 7], by introducing BChl c in an artificial anistropic matrix. An anisotropic matrix, such as a nematic liquid crystal (LC), because of its fluid and oriented structure, has proven to be a good model system for studying the anisotropy parameters of pigment molecules [12 14] and has allowed the investigation of the interaction between close-lying, uniaxially oriented, and uniformly distributed pigment molecules [12,14]. In such a system no chlorophyll-chlorophyll (Chl) aggregation was observed even at high (10 3 M) concentration [12] and the pigment was very stable and occurred only in monomeric form, due to strong interaction with the LC [12,14]. Previously [13,14] the orientation of transition moments (TMs) with respect to the skeleton of the pigment molecule were established for various Chls embedded in LC. In this study the averaged angles between T M of the Q, band of BChl c and the LC orientation axis were established. In LC there are several factors depolarizing emitted fluorescence: torsional vibrations and rotation of pigment molecules, and also excitation energy migration between differently oriented molecules [15-17]. In an anisotropic system these agents perturbe both polarized components of the emission differently; therefore fluorescence anistropy is a function of time after sample excitation. In a case of continuous light excitation and cylindrical symmetry of a p i g m e n t - L C system information about averaged Legendre polynomials ~P2) and ~P4) can be obtained [15]. Previously [13,18-20], when the orientation of Chl a, Chl b and BChl a in the same matrix was investigated, it was found
that various chlorophylls exhibit different (P25 and ( P 4 ) values [18,19]. The absorption, fluorescence and delayed luminescence (DL) spectra have been measured [21]. The circular dichroism (CD) measurements were done because they provide information about uniaxial orientation of pigment in an anisotropic matrix. Previously [12] it was shown that the uniaxial pigment orientation in LC introduces the highest contribution to the measured CD signal because of the low intrinsic chirality of chlorophylls and the small contributions from other elements of the Miiller matrix. In this paper we focus on the interaction of the monomeric form of BChl c with the anisotropic surroundings and, by comparing the behavior of BChl c with that of bacteriopheophytin c (BPhe c), on the influence of Mg on these interactions. The next paper (Part 2) of this series will be concerned with the properties of aggregated forms (dimers and tetramers) of BChl c in the same artificial matrix and the mutual interactions between the pigment molecules.
2. Materials and methods
3~R-8,12 diethyl farnesyl BChl c (R[E,E] BChl cv [22]) was isolated from the green sulfur bacterium P r o s t h e c o c h l o r i s a e s t u a r i i (strain 2 K). The different homologs of BChl c were separated on a normal phase silica H P L C column (Senshupak 5251-N, 250 m m x 20 m m i.d.), cooled to 4°C in an ice-water bath, using a mixture of n-hexane, 2-propanol and methanol (100/3/3, v:v:v) as eluent. The pigments were identified by comparing the elution pattern with the one reported by Smith et al. [23] and by comparing the elution times with those obtained by Otte et al. [24] and van N o o r t et al. [25]. R[E,E] BChl cv was dissolved in chloroform dried over molecular sieves or in water saturated chloroform. BPhe c was prepared by the addition of acetic acid to a solution of BChl c in acetone (pH 2 3). After a 12 h incubation the solvent was evaporated in vacuo and the pigment was dissolved in chloroform. In order to introduce the pigments into the nematic LC the pigment solutions were mixed
A. Dudkowiak et al. / Spectrochimica Acta Part A 52 (1996) 251-264
with an LC matrix solution, and the chloroform was slowly evaporated in vacuo [26]. The concentration of BChl c in the LC was about 5 x 10 3 M. Samples prepared with " d r y " chloroform will be referred to as " d r y " samples; those prepared from chloroform saturated with water as " w e t " samples. Samples containing BPhe c were prepared at concentrations of 5 × 10 4 M and 5 × 10 3 M. The following LC solvents were used: p-methoxy-benzylideno-p'-butylaniline ( MBBA ) / p - ethoxybenzolideno - p ' - butylaniline (EBBA) (3/2, v:v) and 4 - ( t r a n s - 4 ' - n - h e x y l c y c l o hexyl)-isothiocyanato-benzene (6CHBT). The pigmented LC was situated in a cell with windows covered by an orienting layer. A silicon oxide (SiOx) layer was deposited in vacuo to form this orienting layer. As a result of such a deposition the LC molecules were aligned uniaxially within the cell, tilted at an angle of 20 ° to the plane of the windows [12,13]. In good approximation such a cell can be treated as uniaxially oriented with the axis parallel to the plane of the windows because the projection of polarized components on this plane introduced only few percent of error. The reference cell containing the pure LC was of the same construction. The cell thickness was 2 0 / t m . As a result of the strong interaction with the LC, the pigment molecules are oriented by a " g u e s t host" effect [12,13]. Absorption and fluorescence spectra were measured using a single beam spectrophotometer [5,27] which was equipped with a G l a n - T h o m p s o n prism in the case of the polarized measurements. The excitation and fluorescence observation directions were both on the same side of the cell, perpendicular to each other. Four polarized components of fluorescence were measured for the oriented cells VVV, VHV, V H H , VVH (H, horizontal; V, vertical). The first and the last letter refer to the direction of the electric vector of the excitation and fluorescence light, respectively, and the middle letter refers to the position of the orientation axis of the LC matrix. For C D measurements, a photoelastic modulator (PEM FS-4, Hinds International Inc.) operating at 50 kHz, was inserted in front of the sample [5,27]. Separation of the overlapping absorption bands of BChl was done according to the approximation
253
of Thulstrup and co-workers [28,29]. The measured All and A± were inserted in the equations A~.-- All -- d~A ± and A x = A ± - d ° Aii to obtain the absorption reduced components A, and A~., which represent the total sum of the absorption polarized along the main axes of the BChl molecule. The x and y axes go through the s e c o n d - f o u r t h and the first-third pyrrole ring [13,30], respectively, d~ and d ° are so-called "reduction factors" which are chosen in the range 0 to 1 to eliminate the contribution from A± and All, respectively. The angles between the T M s of BChl and the LC orientation axis were also calculated. Delayed luminescence (DL) was measured with an apparatus described elsewhere [31]. The excitation light source was a combination of a nitrogen and a dye laser (type LN1120C/LD2C, PRALaser Inc.). Pulse duration was 200 ps (full width at half m a x i m u m (FWHM)). Signals were gathered in a multichannel analyzer and computer processed. All D L spectra were taken with an additional delay of 200 ns with respect to the laser peak in order to eliminate the p r o m p t flourescence contributions. 3. Results 3.1. Bacteriochlorophyll c in CHCI3
Because the [E,E] BChl CF was introduced to the LC from a chloroform solution, the properties of the pigment is this solvent were also investigated. The absorption spectra, the second derivative of absorption and the C D spectra of BChl c (4.4 Iz M) in dried chloroform are shown in Fig. 1. The main absorption maxima, as determined from the second derivative, are located at 668 nm and 435 nm. The shapes of these spectra indicate that only the monomeric form of the pigment is present in the solution. The abosrption spectrum is similar to the m o n o m e r spectrum of [E,E] BChl cv in acetone reported by Otte et al. [24] and to the spectrum of monomeric 8-n-propyl-12-ethyl farnesyl BChl c ([Pr,E] BChl cv [22]) in dichloromethanemethanol reported by Olson and Cox [32]. It appears that the tendency to form oligomers is much lower for [E,E] BChl Cv than for [Pr, E] BChl c~, where aggregation was already observed at 0.26 /IM in chloroform [33].
254
A. Dudkowiak et al. / Spectrachimica Acta Part A 52 (1996) 251-264
~D
0.8
-0.8
0.6
-0.6
0.4
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.
.
.
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0.2 -:
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.
v
.
400
.
.
.
.
,
500
,
,
600
Wavelength
,
,
n A
~1~1"
700
(rim)
Fig. 1. Absorption (solid line), second derivative of absorption (dotted line) and CD (insert) spectra of BChl c in CHCI~; c = 4.4/~ M; light path 1 cm.
25.0
,--
20.0
~
15.0
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lo.o
0
2
m
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I ~ I I I t I I I t I t I I I I I I ..,,
! ::
-.
- -
exc.435
nm
.......
exc.636
nm
----
exc.668
nm
\\
0.0
600
7OO
Wavelength
80O
(nm)
Fig. 2. Fluorescence spectra of BChl c in CHCI~ at various wavelengths of excitation; c = 4 . 4 / ~ M .
The C D spectrum (insert Fig. 1) has a low amplitude. The spectrum is similar to that reported for monomeric [Pr,E] BChl cF [32]. The sample is isotropic, which means that the C D signal is a measure o f intrinsic chirality o f the pigment (for[E,E] - 9 . 0 m M ~ cm J at 668 nm which is comparable to - 1 1 m M ~ c m - ~ for
[Pr,E] BChl c~ at 664 nm [32]). For Chl a the intrinsic chirality is - 1 3 . 8 m M ~ cm ~ at 657 nm [34]. The shape o f the fluorescence spectra o f the pigment in dry chloroform was independent o f the excitation wavelength (Fig. 2) and had a main m a x i m u m at 675 rim. For monomeric [Pr,E] BChl
A. Dudkowiak et al./' Spectrochimica Acta Part A 52 (1996)251
Cv a fluorescence m a x i m u m at 674 nm has been reported [33]. The solution also showed a strong D L signal with an emission spectrum similar to that of p r o m p t fluorescence (Fig. 3) and similarly independent of the exciation wavelength (results not shown). This shows that D L is emitted from the excited singlet state of the monomeric pigment. Similar features of D L have previously been observed for Chl a in other solvents [31,35]. The ratio of D L yield to the p r o m p t fluorescence yield, calculated from the extrapolated value of the emission intensity during a short time after the laser peak and the intensity of the whole delayed emission, ranged from 0.2 to 0.3 for all measured samples. This value is higher than observed for Chl a in a polymer matrix where it was about 0.15 [35]. In this evaluation the DE intensity is overestimated, and it can be used only for a comparison of the D L to p r o m p t fluorescence intensity ratio in various samples. The decay time r of the D L of BChl c was 17 _+ 2 ps, independent of the excitation wavelength. This is somewhat longer than the D L decay time ( 9 - 1 5 ps) for Chl a in a polyvinyl alcohol water solution [35]. Because the D L of 500 ~,.x-438nm
f
\".....
0
&
m
.E
.........
5.2-10.2ps
---
10.2-15.2ps
:
250 \\\ ;i t J I j : !
2
20.2-25.2ps 45.2-50.2ps
i I
:. ':
: I
'.
:/ , / \ , .
650
---- -
"\
700
-....'
'....
750
800
Wavelength (nm) Fig. 3. Delayed luminescence spectra of BChl c in CHCI3; c= 31 /~M; wavelength of excitation, 438 nm. Time windows are given in the figure.
264
255
BChl c in chloroform exhibits properties similar to those previously observed for Chl a [35], if probably has a similar origin. This means that the D L is generated by the ionization and delayed recombination of pigment molecules [31,35]. Krasnovsky et al. [36] reported that BChl c in rigid matrix (polymethyl methacrylate) emits D L with a decay time of about 1 ms at room temperature. This luminescence was quenched by oxygen and diminished upon lowering the temperature and therefore was supposed to be generated with participation of the triplet state. On the basis of our results we cannot exclude that the observed D L is, at least partially, generated by thermal activation from the triplet to the excited singlet state. 3.2. BChl c in liquid crystals
The polarized and non-polarized absorption spectra of [E,E] BChl cv in the nematic LC matrix are shown in Fig. 4(a) and Fig. 5(a). The absorption parameters of [E,E] BChl cv in organic solvents and in LC matrix are given in Table 1. The absorption spectra of BChl c in nematic solvent were slightly red-shifted with respect to those in "wet" or " d r y " chloroform. This shift was concentration independent (results not shown) and is due to interaction of the pigment with the LC [37]. The amplitude ratio of the Soret and Q,. absorption bands was lower in LC solvents than in chloroform or acetone, and different for the two polarized components. The F W H M of the Q:, band of BChl c in a " d r y " and in a "wet" LC is about 17-18 nm and is comparable to that of the monomeric form in organic solvents. It is quite different from that observed by Uehara and Olson [38] in water-saturated carbon tetrachloride for aggregated forms of several homologs of BChl c. Thus, from the position and the shape of the absorption spectra we conclude that BChl c in the LC is in the monomeric form. Fig. 4(a) and Fig. 5(a) show that BChl c (concentration c = 5 x 10-3 M) in a MBBA/EBBA mixture introduced to this solvent from dry and hydrated chloroform is uniaxially ordered. The degree of orientation S and the average angles ® between the direction of various absorption transition moments (TMs) of BChl c and the LC orien-
256
A. Dudkowiak et al. / Spectrachimica Acta Part A 52 (1996) 251-264
0.8 a
0.6 0.4
"i
< 0.2
0.0
'
400
500
0.4 o
i
600
700
p
b
0.3
0.2
I I I
~
< 0.1
/
~.~
r 0.0 400
t
500
600
}
!1
700
Wavelength (nm) Fig. 4. Absorption spectra of BChl c in "dry" LC (MBBA/EBBA); c = 5 × 10 3 M. (a) Absorption spectra measured with polarized light (parallel component, dotted line; perpendicular component, dashed line) and non-polarized light (solid line). (b) The absorption components obtained by a reduction procedure (see text): A, = A ± - d~A I (solid line) and A,. = All- d~IA± (dashed line).
tation axis were calculated from an analysis of the spectra of Fig. 4(a) and Fig. 5(a) (Table 2). For the BChl c samples the orientation parameter S is higher in the red band region than in the Soret one. The angle between the LC orientation axis and the Q,, TMs calculated for BChl c equals 46 ° for the "wet" sample and about 48 ° for the " d r y " sample (see Fig. 6). An interpretation of the results in the Soret band region is not straightforward because of the overlap of the B, and B,. TMs. It is of interest to compare these results with those obtained with other Chls located in the same matrix. For Chl a in MBBA/EBBA the angle ® for the Q, T M s was about 32 ° and for Chl b about 56 ° [13]. For other Chls the LC axis was always between the T M s of these two main absorption bands [13]. Thus, the Q,, T M of BChl
c is located between the Q,, T M s of Chl a and Chl b as was also found for other Chls [13,14,20]. Hydration of the matrix changes the average inclination angle only slightly. Applying the reduction procedure according to Thulstrup and co-workers [28,29] gave the reduced absorption components shown in Fig. 4(b) and Fig. 5(b). In regions such as the Soret region where there are strongly overlapping bands, the results are difficult to interpet quantitatively, but in the Q, region the spectra indicate a high degree of orientation of BChl c, especially for the "wet" samples, even if one assumes that the 671 nm band has a pure " y " polarization [13]. The shape of the spectra in the Soret region suggests that the B~(0,0) transition is located near 442 nm and the B,(0,0) T M near 418 nm. From the C D spectra measured in the region of
A. Dudkowiak et al. / Spectrochimica Acta Part A 52 (1996) 251-264
a
0.8
257
si
0.6
0.4 <
0.2
0.0 400
500
600
700
500
600
700
0.5
b 0.4
.= 0
0.3
.< 0.1
~
0.(1 400
Wavelength (nm) Fig. 5. Absorption spectra of BChl c in "wet" LC (MBBA/EBBA); c = 5 x 10 ~ M. (a) Absorption spectra measured with polarized light (parallel component, dotted line; perpendicular component, dashed line) and non-polarized light (solid line). (b) The absorption components obtained by a reduction procedure (see text): A~ = A ± - d°A II (solid line) and A~ = A i l - d~A± (dashed line).
Table 1 Absorption parameters of the monomeric form of R[E,E] BChl c~. in solvents (LC-MBBA/EBBA) Solvent
Soret (S) m a x i m u m position n m
Red (Qv) m a x i m u m position n m
Amplitude ratio
F W H M aQ~, band Into]
Remarks
S/Qy
Chloroform Water sat, chloroform Acetone LC "dry ' ' b
435 435
668 668
1.48 1.50
18 17
Fig. 1 Results not shown
433 442
662 671
LC "wet" b
442
671
1.51 1.11 0.94 1.36 1.22 1.07 1.63
(n) ~ (I]) c (±)c (n) c (11) c (±)c
16 18 17 18 17 17 18
(n) c (I])c (±) ~ (n) c (11) c (±)e
Ref. [24] Fig. 4(a)
Fig. 5(a)
a F W H M , full width at half m a x i m u m . b LC " d r y " (or "wet"), BChl c introduced into LC from chloroform (or water saturated chloroform). n, ]], ± are components of absorption: non-polarized, parallel or perpendicular to LC orientation axis, respectively.
258
A. Dudkowiak et al. / Spectrachimica Acta Part A 52 (1996) 251-264
Table 2 Parameters calculated from polarized absorption and emission spectra of BChl c and BPhe c in liquid crystal solvents: (1) MBBA/EBBA, (2) 6CHBT ~ Pigments in LC ~'
Max/nm
S b
O/deg c
Max/nm
(P2) d
(P4) e
BChl c 5 x 10 3 M "dry"
671
0.18
48
678
0.29
-0.04
671
0.22
46
678
0.26
0.48
544 671 553 672
0.06 0.15 0.11 0.12
52 49 50 50
675
0.06
-0.53
675
0.03
- 0.43
(1) BChl c 5 x I0 -3 M "wet"
(1) BPhe c 5 x 10 -4 M "dry" (l) BPhe c 5 × 1 0 3 M "dry" (2)
" "dry" (or "wet") indicates pigments introduced into LC from chloroform (or water saturated chloroform). b S = (AII-A±)/(AII + 2A±). ® is the average angle between the various TMs of absorption and LC orientation axis (on the basis of the formula A :, - A • = 3/2A (3 cos-* 0 - - 1 ) ) where A I and A± are polarized (parallel and perpendicular, respectively) components of absorption. d (P2) e
2 + 7 r ~ - 14rb + 5rarb 2 3 - 14ra + r b - lOrJb" --12 + 21r~ + 21rb-- 3Or~rb 2 3 - 14r~ + rb-- 10rJb
the red a b s o r p t i o n b a n d (Fig. 7) it follows that the CD signal measured for an oriented sample is predominantly dependent on the axial orientation of the cell, i.e. it is related to cell linear dichroism.
LC w i n d o w
The observed CD is two orders of magnitude higher than the intrinsic CD of BChl c (Fig. 1) and changes with rotation of the sample with respect to the CD spectrometer light beam. The CD maximum is located in the region of the pigment absorption band. Fig. 8 shows the polarized fluorescence of BChl c in MBBA/EBBA (8a for the "dry", 8b for the "wet" sample, concentration 5 x 10 3 M). The 10 //,,~ // O
g
.
.
.
~.
x ~ \
/ ./.;'f~'<'5.~"
.
65*
54*
4s.
-5
<
70=48*.
L C axis
J
-10
-15 600
I
650
700
Wavelength (nm) Fig. 6. Uniaxial orientation of BChl c in LC. (LC window, plane of the cell window; E, electric vector of incident light which is parallel to the window plane; LC axis, axis of liquid crystal orientation.)
Fig. 7. Circular dichromism spectra of BChl c in LC (MBBA/ EBBA) "dry" sample taken for various angles between LC orientation axis and vertical direction of the polarizer in the CD apparatus; c = 5 x 10 3 M.
259
A. Dudkowiak et al. I Spectrochimica Acta Part A 52 (1996) 251 264
m a i n fluorescence m a x i m u m is located at 678 nm. Like the a b s o r p t i o n b a n d , it is shifted to somewhat longer wavelength with respect to its position in c h l o r o f o r m (Fig. 2). The spectra were corrected for the different sensitivity o f the app a r a t u s for h o r i z o n t a l l y a n d vertically polarized light. T h e fluorescence was polarized to a high degree (the VVV c o m p o n e n t was m u c h higher t h a n the V H V one). F o r the " d r y " sample, the intensity o f the emission in the p e r p e n d i c u l a r c o m p o n e n t was i n d e p e n d e n t o f the sample rotation (VVH = V H H ) (Fig. 8(a)). This d e m o n s t r a t e s the strong influence of polarized photoselection o n the emission anistropy. The difference between the VVV a n d V H V c o m p o n e n t s shows that the o r i e n t a t i o n o f the sample has a n influence o n the parallel polarized c o m p o n e n t . As usual for a uniaxially oriented sample [39,40], four coefficients o f emission a n i s o t r o p y were calculated. F r o m emission coefficients average values of Legendre polyn o m i a l s (P2) a n d {P4) were calculated [40]. The definitions a n d values o f all the a n i s o t r o p y of emission coefficients, a n d o f the Legendre polyn o m i a l s are s u m m a r i z e d in Tables 2 a n d 3. F o r BChl c values o f {P2) are higher t h a n order p a r a m e t e r s S o b t a i n e d from a b s o r p t i o n spectra (Table 2). It is a n unexpected result, b u t it is possible that as previously [40] the p i g m e n t molecules differently oriented with respect to LC have v a r i o u s yields o f fluorescence. The " w e t "
2.5
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I I
2.0
O
X
xx...+~. vvv
I/
:"'"-.. I .: "...~.I /:"
1.5
oo
// ~
a
i:
1.0 I
..
I:: ~ /" /
~,
VHV
\
"~./VHH ' , VVH
~,"? ............... ,~,
0.5
~~,...~
7;7~Z''',
0.0 690
650 b
2.0 ~ ~ o~
770
r\
II , .jvvv
1.5
#/ .., \~/VHH 1.0
i/i~ \
O
2 m
730
/l:l
vv. \
- \\~.
0.5
.- , . . . . . . . .
0.0
690
650
~~., 730
770
Wave l e n gt h (nm)
Fig. 8. Polarized fluorescence spectra of BChl c in LC (MBBA/ EBBA): (a) "dry" sample, (b) "wet" sample. Excitation wavelength, 440 rim. The polarized components notation is given in the text.
Table 3 Coefficients of pigment fluorescence anisotropy in liquid crystal solvents: (l) MBBA/EBBA, (2) 6CHBT Sample
BChl c 5 × 10-3 M +'dry" ( 1) BChl c 5×10 3 M "'wet" (1) BPhe c 5x 10 4 M "dry" (1) BPhe c 5×10 3 M "dry" (2)
.ie~/nm
2em/nm
Coefficienta ra
Fb
Fc
Fd
420
678 730 678 730 675
0.52 0.29 0.21 0.32 -0.05
0.10 0.21 -0.14 - 0.09 0.02
0.15 0.07 0.16 0.14 -0.10
0.00 0.00 --0.18 -- 0.22 -0.07
420
675
0.00
0.10
--0.11
--0.01
440 440
All coefficients have an accuracy of _+0.01; VVV- VVH r, = VVV + 2VVH;
VHV-- VHH rb -- VHV + 2VHH;
VVV- VHV r,. = VVV+ 2VHV;
Fd
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A. Dudkowiak et al. / Spectrachimica Acta Part A 52 (1996) 251 264 100
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Fig. 9. Delayed luminescence of BChl c in LC (MBBA/EBBA); c = 5 x 10 3 M; time windows as in Fig. 3; wavelengths of excitation (2ex) are given in the figure. (a, b and d) "wet" samples, (c) "dry" sample; (a and b) measured in polarized light (component notation given in text), (c and d) in non-polarized light. Decay time r: (a) 15 + 2 ,us, (b) 17 _+2/zs, (c) 20 _+2 #s, (d)
20+2 ,us. BChl c sample and the BPhe c samples exhibit lower values o f (P2) than o f S (Table 2) which is related to depolarization o f fluorescence. ( P 4 ) is negative and has very high absolute values, m u c h higher than for other Chls [18,19]• ( P 4 ) is very sensitive to m o l e c u l a r m o v e m e n t s [15]. F o r liquid crystal m o l e c u l e s alone [15], ( P 4 ) is negative before the introduction by the correction on rotational depolarization, but it b e c a m e positive after correction. F r o m resonance R a m a n spectra o f Chl
a in LC, ( P 4 ) was calculated for the vibration 1692 c m - 1 (9-keto-carbonyl) [18] and was found to be negative. The direction o f the investigated vibration is close to that o f Qy TM. The m u c h larger absolute values o f negative ( P 4 ) for BChl c than those obatined for Chl a suggests the occurence o f the very strong depolarizing factors for BChl c in LC. F r o m ( / ' 4 ) it is possible to calculate the angle fl between the axis o f molecular s y m m e try and the major axis o f L C orientation [18]. High
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negative values of ( P 4 ) suggest large values of ft. From anistropy of emission coefficients even without calculation of (P4) some qualitative conclusions concerning pigment distribution can be drawn. The anisotropy coefficient r a is high for the samples with a high fraction of molecules oriented at a small angle to the LC orientation axis and having absorption and emission TMs which form a mutually small angle. At the main emission maximum (at 678 nm) ra is higher from the " d r y " sample (5 × 10 3 M) than for the "wet" one. The coefficient re has to be large when the fraction of "well oriented" molecules contributes strongly to the measured emission. In the 730 nm region this coefficient is different for both sample types. It is rather low for the " d r y " sample and high for the "wet". More dramatic effects are observed in the
(c = 5 x 1 0 - 4 M ) a n d (b) 6 C H B T
(c = 5 x 1 0 - 3 M ) ; p a r a l l e l
730 nm region for the coefficient r d which became negative for the "wet" sample, whereas it is zero for the " d r y " sample. This shows that in the "wet" sample the number of molecules oriented at a large angle to the LC axis is higher than the number of well-oriented molecules. For "wet" samples the coefficient rd is also negative in a region of the main emission band (at 678 nm). This shows that the presence of water perturbs the arrangement of the pigment molecules in the LC matrix. This conclusion is supported by the negative values of r b observed for the "wet" sample. This coefficient is negative when molecules oriented at large angles contribute significantly to the emission (molecules with a wide distribution function). Because ® obtained from absorption spectra is similar to that for the other Chls the negative values of anisotropy are due to depolarizing agents. The (P4)
A. Dudkowiak et al./ Spectrachimica Acta Part A 52 (1996) 251-264
262
1.6
a
i
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m a x i m u m (Fig. 8). The position of the band was independent of the excitation wavelength and the light polarization (Fig. 9(a), (b)). Thus, the mechanism of D L generation seems to be the same as for BChl c in chloroform. The perpendicular emission component (VVH) has a higher intensity than the parallel one (VVV). Thus, the D L depends on t he orientation of the emitting molecule with respect to the LC matrix. This shows that the D L depends on the interactions between the LC and the pigment.
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W a v e l e n g t h (nm) Fig. 11. Fluorescence spectra of BPhe c in LCs: (a) MBBA/' EBBA, c = 5 × 1 0 - 4 M ; ( b ) 6CHBT, c = 5 x 10 3 M. Excitation wavelength, 420 nm; polarized notation given in text.
values are in agreement with this discussion showing that the "wet" sample exhibits a wider distribution function than the " d r y " sample. Thus, the presence of water perturbs the arrangement of fluorescent molecules to a higher degree and has a stronger influence on the T M s distribution function around the preferential orienation axis than on their average angle of inclination. This distribution function cannot be obtained from absorption anisotropy, but a change in it can be indicated by different fluorescence anistropy results (Table 3). Fig. 9 shows the D L spectra of [E,E] BChl c~ in the LC. Fig. 9(c) and (d) show that the " d r y " and "wet" samples measured in non-polarized light have similar D L spectra with similar decay times. The spectra show that the D L is predominantly emitted by the monomeric species; the position of the main m a x i m u m is similar to that of the fluorescence
3.3. Bacteriopheophytin c in liquid crystal matrix Fig. 10 shows the polarized absorption spectra of BPhe c in MBBA/EBBA (Fig. 10(a)) and in 6 C H B T (Fig. 10(b)). In Table 2 the absorption parameters calculated from these spectra are presented. The average orientation of the pigment axes appears to be similar to that of a " d r y " sample of BChl c. The type of matrix had little effect on the pigment orientation. The shapes and the magntiude of the C D spectra for BPhe c (not shown), as for BChl c (Fig. 7), were predominantly a result of the pigment orientation. With an angle of 45 ° between the sample axis and the electric vector of light the C D signal had a very low amplitude. The fluorescence spectra of BPhe c (Fig. 11) showed a lower emission polarization than those of BChl c (Fig. 8). The values of the coefficient of emission anisotropy and ( P 4 ) (Table 3) suggest that for the BPhe c molecules the distribution function around the orientation axis is similar to that for BChl c in the "wet" sample (Table 2). BPhe c exhibits stronger depolarization of fluorescence than BChl c because for BPhe c (P=) is much lower than S. The type of LC matrix had only a small influence on the BPhe c emission anisotropy. The difference between the BChl c and BPhe c orientations in the same LC shows the influence of Mg on the L C - p i g m e n t interaction. A similar difference has been found by Wr6bel et al. [41] for Chl a in a polymer matrix. The D L spectra of BPhe c (Fig. 12) showed an emission in the same region where the fluorescence is located. The ratio of the D L and fluorescence intensity was about 0.3, i.e. similar to that for BChl c, and also the D L decay time (r = 16 + 2 / i s ) was the same as that for BChl c.
A. Dudkowiak et al. / Spectrochimica Acta Part A 52 (1996) 251-264
6000
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T h e a u t h o r s w i s h to t h a n k A . H . M . de W i t for c u l t u r i n g t h e b a c t e r i a . A . D . w o u l d like to t h a n k t h e E u r o p e a n Science F o u n d a t i o n f o r a S h o r t t e r m T r a v e l G r a n t . T h e i n v e s t i g a t i o n w a s supp o r t e d by t h e N e t h e r l a n d s Foundation for C h e m i c a l R e s e a r c h ( S O N ) , f i n a n c e d by t h e N e t h e r l a n d s O r g a n i z a t i o n f o r Scientific R e s e a r c h (NWO) and by Poznafi University of Technology g r a n t 62-1 12/2.
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References
- - - - 2 ....
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700
750
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Wavelength (nm) Fig. 12. Delayed luminescence spectra of BPhe c in LC (6CHBT); c = 5 x l0 3 M. Time windows and wavelength of excitation (2ex) are given in the figure. Decay time z = 16 _+2 /~s. Similar decay times were obtained in MBBA/EBBA and for various other wavelengths of excitation.
4. Conclusions (1) In all t h e i n v e s t i g a t e d s a m p l e s t h e m o n o m e r i c species is p r e d o m i n a n t . (2) T h e i n t e r a c t i o n s b e t w e e n the L C a n d t h e p i g m e n t m o l e c u l e s a r e s t r o n g . T h e y c a n be changed by the presence of water molecules. These changes are demonstrated predominantly by a c h a n g e in the p o l a r i z a t i o n o f f l u o r e s c e n c e . T h e c o m p a r i s o n o f the p o l a r i z e d a b s o r p t i o n a n d f l u o r e s c e n c e results s h o w s t h e o c c u r r e n c e o f t h e strong depolarization of emission. (3) T h e t y p e o f L C m a t r i x is n o t v e r y i m p o r t a n t a n d has o n l y a s m a l l i n f l u e n c e o n t h e o r i e n t a t i o n and on the emission anistropy. (4) T h e o r i e n t a t i o n s o f B C h l c a n d B P h e c in the s a m e m a t r i x are similar, b u t the p r e s e n c e o f Mg influences the interactions between LC and pigment molecules, changing the distribution of p i g m e n t in L C .
[1] S.C. Holt, S.F. Conti and R.C. Fuller, J. Bacteriol., 91 (1966) 311. [2] A.J. Hoff and J. Amesz, in H. Scheer (Ed.), Chlorophylls, CRC Press, Boca Raton, 1991, p. 723. [3] S.T. Daurat-Larroque, K. Brew and R.E. Fenna, J. Biol. Chem., 261 (1986) 3607. [4] P.A. Lyle and W.S. Struve, J. Phys. Chem., 94 (1990) 7338. [5] S.C.M. Otte, J.C. van der Heiden, N. Pfennig and J. Amesz, Photosynth. Res., 28 (1991) 77. [6] R.J. van Dorssen, H. Vasmel and J. Amesz, Photosynth. Res., 9 (1986) 33. [7] R.J. van Dorssen, P.D. Gerola, J.M. Olson and J. Amesz, Biochim. Biophys. Acta, 848 (1986) 77. [8] R.E. Blankenship, D.C. Brune and B.P. Wittmershaus, in S.E. Stevens, Jr. and D.A. Bryant (Eds.), Light-Energy Transduction in Photosynthesis. Higher Plants and Bacterial Models, American Society of Plant Physiology, Rockville, MD, 1988, p. 32. [9] J.M. Olson, Biochim. Biophys. Acta, 594 (1980) 33. [10] T. Wechsler, F. Suter, R.C. Fuller and H. Zuber, FEBS Lett., 181 (1985) 173. [11] A.R. Holzwarth and K. Schaffner, Photosynth. Res., 41 (1994) 225. [12] D. Fra~ckowiak, D. Bauman and M.J. Stilman, Biochim. Biophys. Acta, 681 (1982) 273. [13] D. Fr~ckowiak, S. Hotchandani and R.M. Leblanc, Photobiochem. Photobiophys., 6 (1983) 339. [14] D. Fr~tckowiak, D. Bauman, H. Manikowski and T. Martynski, Biophys. Chem., 6 (1977) 369. [15] I.N. Dozov and I.I. Penchev, J. Luminesc., 22 (1980) 69. [16] A. Arcioni, R. Tarroni and C. Zannoni, in B. Samori and E.W. Thulstrup (Eds.), Polarized Spectroscopy of Ordered Systems, Kluwer Academic Publishers, 1988, p. 421. [17] R.E. Dale and R.K. Bauer, Acta Phys. Pol. A, 40 (1971) 853. [18] D. Wr6bel and M. Kozielski, Biophys. Chem., 29 (1987) 309.
264
A. Dudkowiak et al. / Spectrachimica Acta Part A 52 (1996) 251-264
[19] D. Wr6bel and M. Kozielski, Biophys. Chem., 33 (1989) 127. [20] D. Bauman and D. Wr6bel, Biophys. Chem., 12 (1980) 83. [21] D. Fr~ckowiak, B. Zelent, H. Malak, R. Cegielski, J. Goc. M. Niedbalska and A. Ptak, Biophys. Chem., 54 (1995) 95. [22] K.M. Smith, Photosynth. Res., 41 (1994) 23. [23] K.M. Smith, C.W. Craig, L.A. Kehres and N. Pfennig, J. Chromatogr., 281 (1983) 209. [24] S.C.M. Otte, E.J. van de Meent, P.A. van Veelen, A.S. Pundsnes and J. Amesz, Photosynth. Res., 35 (1993) 159. [25] P.I. van Noort, C. Francke, N. Schoumans, S.C.M. Otte, T.J. Aartsma and J. Amesz, Photosynth. Res., 41 (1994) 193. [26] D. Fr~ckowiak and A. Ptak, Photosynthetica, 30 (1994) 553. [27] C. Francke, S.C.M. Otte, J.C. van der Heiden and J. Amesz, Biochim. Biophys. Acta, 1186 (1994) 75. [28] E.W. Thulstrup, J. Michl and J.H. Eggers, J. Phys. Chem., 74 (1970) 3868. [29] J. Michl, E.W. Thulstrup and J.H. Eggers, J. Phys. Chem., 74 (1970) 3878. [30] R. Journeaux and R. Viovy, Photochem. Photobiol., 28 (1978) 243. [31] A. Planner and D. Frgckowiak, Photochem. Photobiol., 54 (1991) 445.
[32] J.M. Olson and R.P. Cox, Photosynth. Res., 30 (1991) 35. [33] J.M. Olson and J.P. Pedersen, Photosynth. Res., 25 (1990) 25. [34] C. Houssier and K. Sauer, J. Am. Chem. Soc., 92 (1970) 779. [35] D. Fra~ckowiak, A. Planner and J. Goc, Photochem. Photobiol., 58 (1993) 737. [36] A.A. Krasnovsky, Jr, P. Cheng, R.E. Blankenship, T.A. Moore and D. Gust, Photochem. Photobiol., 57 (1993) 324. [37] D. Fr~ckowiak, J. Szurkowski, B. Szych, S. Hotchandani and R.M. Leblanc, Photobiochem. Photobiophys., 12 (1986) 9. [38] K. Uehara and J.M. Olson, Photosynth. Res., 33 (1992) 251. [39] D. Fr~ckowiak, A. Dudkowiak, B. Zelent and R.M. Leblanc, J. Fluorescence, 1 (1991) 225. [40] D. Frockowiak, I. Gruda, M. Niedbalska, M. Romanowski and A. Dudkowiak, J. Photochem. Photobiol. A, 54 (1990) 37. [41] D. Wr6bel, M. van Zandvoort, G. van Ginkel and Y.K. Levine, Photosynthetica, 30 (1994) 485.
ELSEVIER
Spectrochimica Acta Part A 52 (1996) 251 264
Spectral properties of bacteriochlorophyll c in nematic liquid crystal. Part 1. Monomeric forms of dye A. D u d k o w i a k a,,, C. F r a n c k e a, J. A m e s z ~, A. Planner b, I. H a n y 2 b, D. F r o c k o w i a k b Department ()[" Biophysics, Huygens Laboratory, University (~["Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands b Moleeular Physies Laboratory, Institute o/' Physics, Poznafi University of Technology Piotrowo 3, 60-965 Poznah, Poland
Received 31 May 1995; accepted in final form 21 September 1995
Abstract
The spectroscopic features of bacteriochlorophyll c and bacteriopheophytin c in a nematic liquid crystal matrix have been investigated. Absorption, circular dichroism, fluorescence and time resolved delayed luminescence spectra have been measured. The pigment is introduced to the liquid crystal from a dry and from a hydrated chloroform solution. In both cases the pigment is in the monomeric form. Hydration of the solvent and the presence or absence of the central Mg atom affect the interaction of the pigment molecules with the liquid crystal matrix, changing the fluorescence anisotropy. A model for the bacteriochlorophyll c orientation in the liquid crystal is proposed and the averaged angles between the transition moments and the liquid crystal orientation axis are determined. A slow process (in the microsecond range) of radiative deactivation of energy absorbed by the pigments is observed. This delayed emission could be due to pigment ionization and delayed charge recombination and/or thermal activation from the triplet to the excited singlet state. Keywords: Bacteriochlorophyll c; Bacteriopheophytin c; Delayed luminescence; Liquid crystal; Polarized spectroscopy
Abbreviations
BChl, b a c t e r i o c h l o r o p h y l l ; [E,E] BChl c v, 8,12-diethyl BChl c esterified with farnesol (F); [Pr,E] BChl Cv, 8 - n - p r o p y l - 1 2 - e t h y l BChl c esterified with farnesol (F); BPhe, b a c t e r i o p h e o p h y t i n ; C D , c i r c u l a r dichroism; 6 C H B T , 4* Corresponding author at Molecular Physics Laboratory, Institute of Physics, Poznafi University of Technology, Piotrowo 3, 60-965 Poznafi, Poland.
(trans-4'-n-hexylcyclohexyl)-isothiocyanato-benz-
ene; Chl, c h l o r o p h y l l ; D L , d e l a y e d luminescence; E B B A , p - e t h o x y b e n z o l i d e n o - p ' - b u t y l a n i l i n e ; F W H M , full w i d t h at h a l f m a x i m u m ; LC, liquid crystal; MBBA, p-methoxy-benzylideno-p'butylaniline; T M , t r a n s i t i o n m o m e n t .
1. I n t r o d u c t i o n
0584-8539/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved S S D I 0584-8539(95)01577-9
B a c t e r i o c h l o r o p h y l l c (BChl
c) is the m a i n
252
A. Dudkowiak et al. / Spectrachimica Acta Part A 52 (1996) 251 264
light-absorbing pigment in various species of green sulfur bacteria [1,2]. It is present in chlorosomes which are attached to the membrane through the F e n n a - M a t t h e w s Olson (FMO) complex [3,4]. BChl c is oriented to a high degree in chlorosomes [5-9]. The spatial arrangement of the pigment molecules, their mutual interactions and their interactions with the chlorosomal proteins have not yet been fully elucidated [9-11]. It thus appeared of interest to study a simpler ordered model simulating some aspects of the properties of the native system. We therefore tried to mimic the natural situation, where the BChls c are oriented in a regular manner [5 7], by introducing BChl c in an artificial anistropic matrix. An anisotropic matrix, such as a nematic liquid crystal (LC), because of its fluid and oriented structure, has proven to be a good model system for studying the anisotropy parameters of pigment molecules [12 14] and has allowed the investigation of the interaction between close-lying, uniaxially oriented, and uniformly distributed pigment molecules [12,14]. In such a system no chlorophyll-chlorophyll (Chl) aggregation was observed even at high (10 3 M) concentration [12] and the pigment was very stable and occurred only in monomeric form, due to strong interaction with the LC [12,14]. Previously [13,14] the orientation of transition moments (TMs) with respect to the skeleton of the pigment molecule were established for various Chls embedded in LC. In this study the averaged angles between T M of the Q, band of BChl c and the LC orientation axis were established. In LC there are several factors depolarizing emitted fluorescence: torsional vibrations and rotation of pigment molecules, and also excitation energy migration between differently oriented molecules [15-17]. In an anisotropic system these agents perturbe both polarized components of the emission differently; therefore fluorescence anistropy is a function of time after sample excitation. In a case of continuous light excitation and cylindrical symmetry of a p i g m e n t - L C system information about averaged Legendre polynomials ~P2) and ~P4) can be obtained [15]. Previously [13,18-20], when the orientation of Chl a, Chl b and BChl a in the same matrix was investigated, it was found
that various chlorophylls exhibit different (P25 and ( P 4 ) values [18,19]. The absorption, fluorescence and delayed luminescence (DL) spectra have been measured [21]. The circular dichroism (CD) measurements were done because they provide information about uniaxial orientation of pigment in an anisotropic matrix. Previously [12] it was shown that the uniaxial pigment orientation in LC introduces the highest contribution to the measured CD signal because of the low intrinsic chirality of chlorophylls and the small contributions from other elements of the Miiller matrix. In this paper we focus on the interaction of the monomeric form of BChl c with the anisotropic surroundings and, by comparing the behavior of BChl c with that of bacteriopheophytin c (BPhe c), on the influence of Mg on these interactions. The next paper (Part 2) of this series will be concerned with the properties of aggregated forms (dimers and tetramers) of BChl c in the same artificial matrix and the mutual interactions between the pigment molecules.
2. Materials and methods
3~R-8,12 diethyl farnesyl BChl c (R[E,E] BChl cv [22]) was isolated from the green sulfur bacterium P r o s t h e c o c h l o r i s a e s t u a r i i (strain 2 K). The different homologs of BChl c were separated on a normal phase silica H P L C column (Senshupak 5251-N, 250 m m x 20 m m i.d.), cooled to 4°C in an ice-water bath, using a mixture of n-hexane, 2-propanol and methanol (100/3/3, v:v:v) as eluent. The pigments were identified by comparing the elution pattern with the one reported by Smith et al. [23] and by comparing the elution times with those obtained by Otte et al. [24] and van N o o r t et al. [25]. R[E,E] BChl cv was dissolved in chloroform dried over molecular sieves or in water saturated chloroform. BPhe c was prepared by the addition of acetic acid to a solution of BChl c in acetone (pH 2 3). After a 12 h incubation the solvent was evaporated in vacuo and the pigment was dissolved in chloroform. In order to introduce the pigments into the nematic LC the pigment solutions were mixed
A. Dudkowiak et al. / Spectrochimica Acta Part A 52 (1996) 251-264
with an LC matrix solution, and the chloroform was slowly evaporated in vacuo [26]. The concentration of BChl c in the LC was about 5 x 10 3 M. Samples prepared with " d r y " chloroform will be referred to as " d r y " samples; those prepared from chloroform saturated with water as " w e t " samples. Samples containing BPhe c were prepared at concentrations of 5 × 10 4 M and 5 × 10 3 M. The following LC solvents were used: p-methoxy-benzylideno-p'-butylaniline ( MBBA ) / p - ethoxybenzolideno - p ' - butylaniline (EBBA) (3/2, v:v) and 4 - ( t r a n s - 4 ' - n - h e x y l c y c l o hexyl)-isothiocyanato-benzene (6CHBT). The pigmented LC was situated in a cell with windows covered by an orienting layer. A silicon oxide (SiOx) layer was deposited in vacuo to form this orienting layer. As a result of such a deposition the LC molecules were aligned uniaxially within the cell, tilted at an angle of 20 ° to the plane of the windows [12,13]. In good approximation such a cell can be treated as uniaxially oriented with the axis parallel to the plane of the windows because the projection of polarized components on this plane introduced only few percent of error. The reference cell containing the pure LC was of the same construction. The cell thickness was 2 0 / t m . As a result of the strong interaction with the LC, the pigment molecules are oriented by a " g u e s t host" effect [12,13]. Absorption and fluorescence spectra were measured using a single beam spectrophotometer [5,27] which was equipped with a G l a n - T h o m p s o n prism in the case of the polarized measurements. The excitation and fluorescence observation directions were both on the same side of the cell, perpendicular to each other. Four polarized components of fluorescence were measured for the oriented cells VVV, VHV, V H H , VVH (H, horizontal; V, vertical). The first and the last letter refer to the direction of the electric vector of the excitation and fluorescence light, respectively, and the middle letter refers to the position of the orientation axis of the LC matrix. For C D measurements, a photoelastic modulator (PEM FS-4, Hinds International Inc.) operating at 50 kHz, was inserted in front of the sample [5,27]. Separation of the overlapping absorption bands of BChl was done according to the approximation
253
of Thulstrup and co-workers [28,29]. The measured All and A± were inserted in the equations A~.-- All -- d~A ± and A x = A ± - d ° Aii to obtain the absorption reduced components A, and A~., which represent the total sum of the absorption polarized along the main axes of the BChl molecule. The x and y axes go through the s e c o n d - f o u r t h and the first-third pyrrole ring [13,30], respectively, d~ and d ° are so-called "reduction factors" which are chosen in the range 0 to 1 to eliminate the contribution from A± and All, respectively. The angles between the T M s of BChl and the LC orientation axis were also calculated. Delayed luminescence (DL) was measured with an apparatus described elsewhere [31]. The excitation light source was a combination of a nitrogen and a dye laser (type LN1120C/LD2C, PRALaser Inc.). Pulse duration was 200 ps (full width at half m a x i m u m (FWHM)). Signals were gathered in a multichannel analyzer and computer processed. All D L spectra were taken with an additional delay of 200 ns with respect to the laser peak in order to eliminate the p r o m p t flourescence contributions. 3. Results 3.1. Bacteriochlorophyll c in CHCI3
Because the [E,E] BChl CF was introduced to the LC from a chloroform solution, the properties of the pigment is this solvent were also investigated. The absorption spectra, the second derivative of absorption and the C D spectra of BChl c (4.4 Iz M) in dried chloroform are shown in Fig. 1. The main absorption maxima, as determined from the second derivative, are located at 668 nm and 435 nm. The shapes of these spectra indicate that only the monomeric form of the pigment is present in the solution. The abosrption spectrum is similar to the m o n o m e r spectrum of [E,E] BChl cv in acetone reported by Otte et al. [24] and to the spectrum of monomeric 8-n-propyl-12-ethyl farnesyl BChl c ([Pr,E] BChl cv [22]) in dichloromethanemethanol reported by Olson and Cox [32]. It appears that the tendency to form oligomers is much lower for [E,E] BChl Cv than for [Pr, E] BChl c~, where aggregation was already observed at 0.26 /IM in chloroform [33].
254
A. Dudkowiak et al. / Spectrachimica Acta Part A 52 (1996) 251-264
~D
0.8
-0.8
0.6
-0.6
0.4
~ -o.o$ .
~ .
.
.
.
'°° O t,O
<
.
,
.
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'"
-0.4
~i
t~
0.2 0.0
..:. . . . . . . .
"":.i
..
-.. i i -" .. : _/ :~/:
...
.>
-0.2
-0.2
~
0.0
" &
0.2 -:
. h A
.
v
.
400
.
.
.
.
,
500
,
,
600
Wavelength
,
,
n A
~1~1"
700
(rim)
Fig. 1. Absorption (solid line), second derivative of absorption (dotted line) and CD (insert) spectra of BChl c in CHCI~; c = 4.4/~ M; light path 1 cm.
25.0
,--
20.0
~
15.0
~
lo.o
0
2
m
5.0
I ~ I I I t I I I t I t I I I I I I ..,,
! ::
-.
- -
exc.435
nm
.......
exc.636
nm
----
exc.668
nm
\\
0.0
600
7OO
Wavelength
80O
(nm)
Fig. 2. Fluorescence spectra of BChl c in CHCI~ at various wavelengths of excitation; c = 4 . 4 / ~ M .
The C D spectrum (insert Fig. 1) has a low amplitude. The spectrum is similar to that reported for monomeric [Pr,E] BChl cF [32]. The sample is isotropic, which means that the C D signal is a measure o f intrinsic chirality o f the pigment (for[E,E] - 9 . 0 m M ~ cm J at 668 nm which is comparable to - 1 1 m M ~ c m - ~ for
[Pr,E] BChl c~ at 664 nm [32]). For Chl a the intrinsic chirality is - 1 3 . 8 m M ~ cm ~ at 657 nm [34]. The shape o f the fluorescence spectra o f the pigment in dry chloroform was independent o f the excitation wavelength (Fig. 2) and had a main m a x i m u m at 675 rim. For monomeric [Pr,E] BChl
A. Dudkowiak et al./' Spectrochimica Acta Part A 52 (1996)251
Cv a fluorescence m a x i m u m at 674 nm has been reported [33]. The solution also showed a strong D L signal with an emission spectrum similar to that of p r o m p t fluorescence (Fig. 3) and similarly independent of the exciation wavelength (results not shown). This shows that D L is emitted from the excited singlet state of the monomeric pigment. Similar features of D L have previously been observed for Chl a in other solvents [31,35]. The ratio of D L yield to the p r o m p t fluorescence yield, calculated from the extrapolated value of the emission intensity during a short time after the laser peak and the intensity of the whole delayed emission, ranged from 0.2 to 0.3 for all measured samples. This value is higher than observed for Chl a in a polymer matrix where it was about 0.15 [35]. In this evaluation the DE intensity is overestimated, and it can be used only for a comparison of the D L to p r o m p t fluorescence intensity ratio in various samples. The decay time r of the D L of BChl c was 17 _+ 2 ps, independent of the excitation wavelength. This is somewhat longer than the D L decay time ( 9 - 1 5 ps) for Chl a in a polyvinyl alcohol water solution [35]. Because the D L of 500 ~,.x-438nm
f
\".....
0
&
m
.E
.........
5.2-10.2ps
---
10.2-15.2ps
:
250 \\\ ;i t J I j : !
2
20.2-25.2ps 45.2-50.2ps
i I
:. ':
: I
'.
:/ , / \ , .
650
---- -
"\
700
-....'
'....
750
800
Wavelength (nm) Fig. 3. Delayed luminescence spectra of BChl c in CHCI3; c= 31 /~M; wavelength of excitation, 438 nm. Time windows are given in the figure.
264
255
BChl c in chloroform exhibits properties similar to those previously observed for Chl a [35], if probably has a similar origin. This means that the D L is generated by the ionization and delayed recombination of pigment molecules [31,35]. Krasnovsky et al. [36] reported that BChl c in rigid matrix (polymethyl methacrylate) emits D L with a decay time of about 1 ms at room temperature. This luminescence was quenched by oxygen and diminished upon lowering the temperature and therefore was supposed to be generated with participation of the triplet state. On the basis of our results we cannot exclude that the observed D L is, at least partially, generated by thermal activation from the triplet to the excited singlet state. 3.2. BChl c in liquid crystals
The polarized and non-polarized absorption spectra of [E,E] BChl cv in the nematic LC matrix are shown in Fig. 4(a) and Fig. 5(a). The absorption parameters of [E,E] BChl cv in organic solvents and in LC matrix are given in Table 1. The absorption spectra of BChl c in nematic solvent were slightly red-shifted with respect to those in "wet" or " d r y " chloroform. This shift was concentration independent (results not shown) and is due to interaction of the pigment with the LC [37]. The amplitude ratio of the Soret and Q,. absorption bands was lower in LC solvents than in chloroform or acetone, and different for the two polarized components. The F W H M of the Q:, band of BChl c in a " d r y " and in a "wet" LC is about 17-18 nm and is comparable to that of the monomeric form in organic solvents. It is quite different from that observed by Uehara and Olson [38] in water-saturated carbon tetrachloride for aggregated forms of several homologs of BChl c. Thus, from the position and the shape of the absorption spectra we conclude that BChl c in the LC is in the monomeric form. Fig. 4(a) and Fig. 5(a) show that BChl c (concentration c = 5 x 10-3 M) in a MBBA/EBBA mixture introduced to this solvent from dry and hydrated chloroform is uniaxially ordered. The degree of orientation S and the average angles ® between the direction of various absorption transition moments (TMs) of BChl c and the LC orien-
256
A. Dudkowiak et al. / Spectrachimica Acta Part A 52 (1996) 251-264
0.8 a
0.6 0.4
"i
< 0.2
0.0
'
400
500
0.4 o
i
600
700
p
b
0.3
0.2
I I I
~
< 0.1
/
~.~
r 0.0 400
t
500
600
}
!1
700
Wavelength (nm) Fig. 4. Absorption spectra of BChl c in "dry" LC (MBBA/EBBA); c = 5 × 10 3 M. (a) Absorption spectra measured with polarized light (parallel component, dotted line; perpendicular component, dashed line) and non-polarized light (solid line). (b) The absorption components obtained by a reduction procedure (see text): A, = A ± - d~A I (solid line) and A,. = All- d~IA± (dashed line).
tation axis were calculated from an analysis of the spectra of Fig. 4(a) and Fig. 5(a) (Table 2). For the BChl c samples the orientation parameter S is higher in the red band region than in the Soret one. The angle between the LC orientation axis and the Q,, TMs calculated for BChl c equals 46 ° for the "wet" sample and about 48 ° for the " d r y " sample (see Fig. 6). An interpretation of the results in the Soret band region is not straightforward because of the overlap of the B, and B,. TMs. It is of interest to compare these results with those obtained with other Chls located in the same matrix. For Chl a in MBBA/EBBA the angle ® for the Q, T M s was about 32 ° and for Chl b about 56 ° [13]. For other Chls the LC axis was always between the T M s of these two main absorption bands [13]. Thus, the Q,, T M of BChl
c is located between the Q,, T M s of Chl a and Chl b as was also found for other Chls [13,14,20]. Hydration of the matrix changes the average inclination angle only slightly. Applying the reduction procedure according to Thulstrup and co-workers [28,29] gave the reduced absorption components shown in Fig. 4(b) and Fig. 5(b). In regions such as the Soret region where there are strongly overlapping bands, the results are difficult to interpet quantitatively, but in the Q, region the spectra indicate a high degree of orientation of BChl c, especially for the "wet" samples, even if one assumes that the 671 nm band has a pure " y " polarization [13]. The shape of the spectra in the Soret region suggests that the B~(0,0) transition is located near 442 nm and the B,(0,0) T M near 418 nm. From the C D spectra measured in the region of
A. Dudkowiak et al. / Spectrochimica Acta Part A 52 (1996) 251-264
a
0.8
257
si
0.6
0.4 <
0.2
0.0 400
500
600
700
500
600
700
0.5
b 0.4
.= 0
0.3
.< 0.1
~
0.(1 400
Wavelength (nm) Fig. 5. Absorption spectra of BChl c in "wet" LC (MBBA/EBBA); c = 5 x 10 ~ M. (a) Absorption spectra measured with polarized light (parallel component, dotted line; perpendicular component, dashed line) and non-polarized light (solid line). (b) The absorption components obtained by a reduction procedure (see text): A~ = A ± - d°A II (solid line) and A~ = A i l - d~A± (dashed line).
Table 1 Absorption parameters of the monomeric form of R[E,E] BChl c~. in solvents (LC-MBBA/EBBA) Solvent
Soret (S) m a x i m u m position n m
Red (Qv) m a x i m u m position n m
Amplitude ratio
F W H M aQ~, band Into]
Remarks
S/Qy
Chloroform Water sat, chloroform Acetone LC "dry ' ' b
435 435
668 668
1.48 1.50
18 17
Fig. 1 Results not shown
433 442
662 671
LC "wet" b
442
671
1.51 1.11 0.94 1.36 1.22 1.07 1.63
(n) ~ (I]) c (±)c (n) c (11) c (±)c
16 18 17 18 17 17 18
(n) c (I])c (±) ~ (n) c (11) c (±)e
Ref. [24] Fig. 4(a)
Fig. 5(a)
a F W H M , full width at half m a x i m u m . b LC " d r y " (or "wet"), BChl c introduced into LC from chloroform (or water saturated chloroform). n, ]], ± are components of absorption: non-polarized, parallel or perpendicular to LC orientation axis, respectively.
258
A. Dudkowiak et al. / Spectrachimica Acta Part A 52 (1996) 251-264
Table 2 Parameters calculated from polarized absorption and emission spectra of BChl c and BPhe c in liquid crystal solvents: (1) MBBA/EBBA, (2) 6CHBT ~ Pigments in LC ~'
Max/nm
S b
O/deg c
Max/nm
(P2) d
(P4) e
BChl c 5 x 10 3 M "dry"
671
0.18
48
678
0.29
-0.04
671
0.22
46
678
0.26
0.48
544 671 553 672
0.06 0.15 0.11 0.12
52 49 50 50
675
0.06
-0.53
675
0.03
- 0.43
(1) BChl c 5 x I0 -3 M "wet"
(1) BPhe c 5 x 10 -4 M "dry" (l) BPhe c 5 × 1 0 3 M "dry" (2)
" "dry" (or "wet") indicates pigments introduced into LC from chloroform (or water saturated chloroform). b S = (AII-A±)/(AII + 2A±). ® is the average angle between the various TMs of absorption and LC orientation axis (on the basis of the formula A :, - A • = 3/2A (3 cos-* 0 - - 1 ) ) where A I and A± are polarized (parallel and perpendicular, respectively) components of absorption. d (P2) e
2 + 7 r ~ - 14rb + 5rarb 2 3 - 14ra + r b - lOrJb" --12 + 21r~ + 21rb-- 3Or~rb 2 3 - 14r~ + rb-- 10rJb
the red a b s o r p t i o n b a n d (Fig. 7) it follows that the CD signal measured for an oriented sample is predominantly dependent on the axial orientation of the cell, i.e. it is related to cell linear dichroism.
LC w i n d o w
The observed CD is two orders of magnitude higher than the intrinsic CD of BChl c (Fig. 1) and changes with rotation of the sample with respect to the CD spectrometer light beam. The CD maximum is located in the region of the pigment absorption band. Fig. 8 shows the polarized fluorescence of BChl c in MBBA/EBBA (8a for the "dry", 8b for the "wet" sample, concentration 5 x 10 3 M). The 10 //,,~ // O
g
.
.
.
~.
x ~ \
/ ./.;'f~'<'5.~"
.
65*
54*
4s.
-5
<
70=48*.
L C axis
J
-10
-15 600
I
650
700
Wavelength (nm) Fig. 6. Uniaxial orientation of BChl c in LC. (LC window, plane of the cell window; E, electric vector of incident light which is parallel to the window plane; LC axis, axis of liquid crystal orientation.)
Fig. 7. Circular dichromism spectra of BChl c in LC (MBBA/ EBBA) "dry" sample taken for various angles between LC orientation axis and vertical direction of the polarizer in the CD apparatus; c = 5 x 10 3 M.
259
A. Dudkowiak et al. I Spectrochimica Acta Part A 52 (1996) 251 264
m a i n fluorescence m a x i m u m is located at 678 nm. Like the a b s o r p t i o n b a n d , it is shifted to somewhat longer wavelength with respect to its position in c h l o r o f o r m (Fig. 2). The spectra were corrected for the different sensitivity o f the app a r a t u s for h o r i z o n t a l l y a n d vertically polarized light. T h e fluorescence was polarized to a high degree (the VVV c o m p o n e n t was m u c h higher t h a n the V H V one). F o r the " d r y " sample, the intensity o f the emission in the p e r p e n d i c u l a r c o m p o n e n t was i n d e p e n d e n t o f the sample rotation (VVH = V H H ) (Fig. 8(a)). This d e m o n s t r a t e s the strong influence of polarized photoselection o n the emission anistropy. The difference between the VVV a n d V H V c o m p o n e n t s shows that the o r i e n t a t i o n o f the sample has a n influence o n the parallel polarized c o m p o n e n t . As usual for a uniaxially oriented sample [39,40], four coefficients o f emission a n i s o t r o p y were calculated. F r o m emission coefficients average values of Legendre polyn o m i a l s (P2) a n d {P4) were calculated [40]. The definitions a n d values o f all the a n i s o t r o p y of emission coefficients, a n d o f the Legendre polyn o m i a l s are s u m m a r i z e d in Tables 2 a n d 3. F o r BChl c values o f {P2) are higher t h a n order p a r a m e t e r s S o b t a i n e d from a b s o r p t i o n spectra (Table 2). It is a n unexpected result, b u t it is possible that as previously [40] the p i g m e n t molecules differently oriented with respect to LC have v a r i o u s yields o f fluorescence. The " w e t "
2.5
""
I I
2.0
O
X
xx...+~. vvv
I/
:"'"-.. I .: "...~.I /:"
1.5
oo
// ~
a
i:
1.0 I
..
I:: ~ /" /
~,
VHV
\
"~./VHH ' , VVH
~,"? ............... ,~,
0.5
~~,...~
7;7~Z''',
0.0 690
650 b
2.0 ~ ~ o~
770
r\
II , .jvvv
1.5
#/ .., \~/VHH 1.0
i/i~ \
O
2 m
730
/l:l
vv. \
- \\~.
0.5
.- , . . . . . . . .
0.0
690
650
~~., 730
770
Wave l e n gt h (nm)
Fig. 8. Polarized fluorescence spectra of BChl c in LC (MBBA/ EBBA): (a) "dry" sample, (b) "wet" sample. Excitation wavelength, 440 rim. The polarized components notation is given in the text.
Table 3 Coefficients of pigment fluorescence anisotropy in liquid crystal solvents: (l) MBBA/EBBA, (2) 6CHBT Sample
BChl c 5 × 10-3 M +'dry" ( 1) BChl c 5×10 3 M "'wet" (1) BPhe c 5x 10 4 M "dry" (1) BPhe c 5×10 3 M "dry" (2)
.ie~/nm
2em/nm
Coefficienta ra
Fb
Fc
Fd
420
678 730 678 730 675
0.52 0.29 0.21 0.32 -0.05
0.10 0.21 -0.14 - 0.09 0.02
0.15 0.07 0.16 0.14 -0.10
0.00 0.00 --0.18 -- 0.22 -0.07
420
675
0.00
0.10
--0.11
--0.01
440 440
All coefficients have an accuracy of _+0.01; VVV- VVH r, = VVV + 2VVH;
VHV-- VHH rb -- VHV + 2VHH;
VVV- VHV r,. = VVV+ 2VHV;
Fd
VVH - VHH VVH + 2VHH
260
A. Dudkowiak et al. / Spectrachimica Acta Part A 52 (1996) 251 264 100
2OO
8
~. :
J~,,-420mn
~t.-41ems
.
WH
VVV
g o
J
el
t~ 50
: /x i
\\::...
iI
I) II
;,a,,
N
ca
.
....
8.2.10.2lJa
~ -
10.2-15.2ps
--
20.2-2s.2p,
--
.... 100
8.2-10.2p8
• /~
-
10.2-15.21~s 20.2-25.21:8
// ~ii'
~.2-.~.
- -
46.2-50.21~8
\\
N'..
~'..
..'",.
0
650
700
750
800
650
700
Wavelength (rim) C
750
800
Wavelength (rim)
d
-
2
k.,~m
800
800 •
°
oo
o
v .....
..
* 10.2-15.21Je "
,oo
[ i ~\ i
~
:
20.2-25.2lJe
- - ,~.2-so.~,
•
i
i \
\'.
..... -.
700
750
i/
J/
5.2-10.2U8 10.2-111.2ps
!
~
2o.2-2s.2po
\i %:. \ " . . . - .... .-.....
...
0
0
650
.
4O0
;I "1
.... ~ -
6.2-10.21Je
800
Wavelength (-,-)
650
700
750
800
Wavelength (~,,,)
Fig. 9. Delayed luminescence of BChl c in LC (MBBA/EBBA); c = 5 x 10 3 M; time windows as in Fig. 3; wavelengths of excitation (2ex) are given in the figure. (a, b and d) "wet" samples, (c) "dry" sample; (a and b) measured in polarized light (component notation given in text), (c and d) in non-polarized light. Decay time r: (a) 15 + 2 ,us, (b) 17 _+2/zs, (c) 20 _+2 #s, (d)
20+2 ,us. BChl c sample and the BPhe c samples exhibit lower values o f (P2) than o f S (Table 2) which is related to depolarization o f fluorescence. ( P 4 ) is negative and has very high absolute values, m u c h higher than for other Chls [18,19]• ( P 4 ) is very sensitive to m o l e c u l a r m o v e m e n t s [15]. F o r liquid crystal m o l e c u l e s alone [15], ( P 4 ) is negative before the introduction by the correction on rotational depolarization, but it b e c a m e positive after correction. F r o m resonance R a m a n spectra o f Chl
a in LC, ( P 4 ) was calculated for the vibration 1692 c m - 1 (9-keto-carbonyl) [18] and was found to be negative. The direction o f the investigated vibration is close to that o f Qy TM. The m u c h larger absolute values o f negative ( P 4 ) for BChl c than those obatined for Chl a suggests the occurence o f the very strong depolarizing factors for BChl c in LC. F r o m ( / ' 4 ) it is possible to calculate the angle fl between the axis o f molecular s y m m e try and the major axis o f L C orientation [18]. High
i-':
A . D u d k o w i a k et al. / S p e c t r o c h i m i c a A c t a P a r t A 5 2 ( 1 9 9 6 ) 2 5 1 - 2 6 4
0.3
261
a
/
0.2
x.".
~
\"...
0.0
~..~.
/
-.;/ "~.
,.., ~ ~
..z~"
~..
i
400
500
600
700
...';
2.0
F\:: 1.5
/
v
1.0 li
<
:! x~ is I! " t"
t!
|-
0.5 ~.. ~'. . . . . . . .
0.0 400
i
......
~ <..
"" ~',."..~,
i ~l ~.22""-----{"
i
~-
500
./
\" . . . . . . i
•
i "-.'.i~ ..... i'.>--'<.'.~,.~.
600
~-
5
/
i
k. i
~.
700
Wavelength (nm) Fig. 10. P o l a r i z e d a b s o r p t i o n s p e c t r a o f B P h e c LCs: (a) M B B A / E B B A c o m p o n e n t , d o t t e d line; p e r p e n d i c u l a r c o m p o n e n t , d a s h e d line.
negative values of ( P 4 ) suggest large values of ft. From anistropy of emission coefficients even without calculation of (P4) some qualitative conclusions concerning pigment distribution can be drawn. The anisotropy coefficient r a is high for the samples with a high fraction of molecules oriented at a small angle to the LC orientation axis and having absorption and emission TMs which form a mutually small angle. At the main emission maximum (at 678 nm) ra is higher from the " d r y " sample (5 × 10 3 M) than for the "wet" one. The coefficient re has to be large when the fraction of "well oriented" molecules contributes strongly to the measured emission. In the 730 nm region this coefficient is different for both sample types. It is rather low for the " d r y " sample and high for the "wet". More dramatic effects are observed in the
(c = 5 x 1 0 - 4 M ) a n d (b) 6 C H B T
(c = 5 x 1 0 - 3 M ) ; p a r a l l e l
730 nm region for the coefficient r d which became negative for the "wet" sample, whereas it is zero for the " d r y " sample. This shows that in the "wet" sample the number of molecules oriented at a large angle to the LC axis is higher than the number of well-oriented molecules. For "wet" samples the coefficient rd is also negative in a region of the main emission band (at 678 nm). This shows that the presence of water perturbs the arrangement of the pigment molecules in the LC matrix. This conclusion is supported by the negative values of r b observed for the "wet" sample. This coefficient is negative when molecules oriented at large angles contribute significantly to the emission (molecules with a wide distribution function). Because ® obtained from absorption spectra is similar to that for the other Chls the negative values of anisotropy are due to depolarizing agents. The (P4)
A. Dudkowiak et al./ Spectrachimica Acta Part A 52 (1996) 251-264
262
1.6
a
i
"-/
"
m a x i m u m (Fig. 8). The position of the band was independent of the excitation wavelength and the light polarization (Fig. 9(a), (b)). Thus, the mechanism of D L generation seems to be the same as for BChl c in chloroform. The perpendicular emission component (VVH) has a higher intensity than the parallel one (VVV). Thus, the D L depends on t he orientation of the emitting molecule with respect to the LC matrix. This shows that the D L depends on the interactions between the LC and the pigment.
VHV VHH
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W a v e l e n g t h (nm) Fig. 11. Fluorescence spectra of BPhe c in LCs: (a) MBBA/' EBBA, c = 5 × 1 0 - 4 M ; ( b ) 6CHBT, c = 5 x 10 3 M. Excitation wavelength, 420 nm; polarized notation given in text.
values are in agreement with this discussion showing that the "wet" sample exhibits a wider distribution function than the " d r y " sample. Thus, the presence of water perturbs the arrangement of fluorescent molecules to a higher degree and has a stronger influence on the T M s distribution function around the preferential orienation axis than on their average angle of inclination. This distribution function cannot be obtained from absorption anisotropy, but a change in it can be indicated by different fluorescence anistropy results (Table 3). Fig. 9 shows the D L spectra of [E,E] BChl c~ in the LC. Fig. 9(c) and (d) show that the " d r y " and "wet" samples measured in non-polarized light have similar D L spectra with similar decay times. The spectra show that the D L is predominantly emitted by the monomeric species; the position of the main m a x i m u m is similar to that of the fluorescence
3.3. Bacteriopheophytin c in liquid crystal matrix Fig. 10 shows the polarized absorption spectra of BPhe c in MBBA/EBBA (Fig. 10(a)) and in 6 C H B T (Fig. 10(b)). In Table 2 the absorption parameters calculated from these spectra are presented. The average orientation of the pigment axes appears to be similar to that of a " d r y " sample of BChl c. The type of matrix had little effect on the pigment orientation. The shapes and the magntiude of the C D spectra for BPhe c (not shown), as for BChl c (Fig. 7), were predominantly a result of the pigment orientation. With an angle of 45 ° between the sample axis and the electric vector of light the C D signal had a very low amplitude. The fluorescence spectra of BPhe c (Fig. 11) showed a lower emission polarization than those of BChl c (Fig. 8). The values of the coefficient of emission anisotropy and ( P 4 ) (Table 3) suggest that for the BPhe c molecules the distribution function around the orientation axis is similar to that for BChl c in the "wet" sample (Table 2). BPhe c exhibits stronger depolarization of fluorescence than BChl c because for BPhe c (P=) is much lower than S. The type of LC matrix had only a small influence on the BPhe c emission anisotropy. The difference between the BChl c and BPhe c orientations in the same LC shows the influence of Mg on the L C - p i g m e n t interaction. A similar difference has been found by Wr6bel et al. [41] for Chl a in a polymer matrix. The D L spectra of BPhe c (Fig. 12) showed an emission in the same region where the fluorescence is located. The ratio of the D L and fluorescence intensity was about 0.3, i.e. similar to that for BChl c, and also the D L decay time (r = 16 + 2 / i s ) was the same as that for BChl c.
A. Dudkowiak et al. / Spectrochimica Acta Part A 52 (1996) 251-264
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Acknowledgments X.x-438nm
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T h e a u t h o r s w i s h to t h a n k A . H . M . de W i t for c u l t u r i n g t h e b a c t e r i a . A . D . w o u l d like to t h a n k t h e E u r o p e a n Science F o u n d a t i o n f o r a S h o r t t e r m T r a v e l G r a n t . T h e i n v e s t i g a t i o n w a s supp o r t e d by t h e N e t h e r l a n d s Foundation for C h e m i c a l R e s e a r c h ( S O N ) , f i n a n c e d by t h e N e t h e r l a n d s O r g a n i z a t i o n f o r Scientific R e s e a r c h (NWO) and by Poznafi University of Technology g r a n t 62-1 12/2.
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References
- - - - 2 ....
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700
750
800
Wavelength (nm) Fig. 12. Delayed luminescence spectra of BPhe c in LC (6CHBT); c = 5 x l0 3 M. Time windows and wavelength of excitation (2ex) are given in the figure. Decay time z = 16 _+2 /~s. Similar decay times were obtained in MBBA/EBBA and for various other wavelengths of excitation.
4. Conclusions (1) In all t h e i n v e s t i g a t e d s a m p l e s t h e m o n o m e r i c species is p r e d o m i n a n t . (2) T h e i n t e r a c t i o n s b e t w e e n the L C a n d t h e p i g m e n t m o l e c u l e s a r e s t r o n g . T h e y c a n be changed by the presence of water molecules. These changes are demonstrated predominantly by a c h a n g e in the p o l a r i z a t i o n o f f l u o r e s c e n c e . T h e c o m p a r i s o n o f the p o l a r i z e d a b s o r p t i o n a n d f l u o r e s c e n c e results s h o w s t h e o c c u r r e n c e o f t h e strong depolarization of emission. (3) T h e t y p e o f L C m a t r i x is n o t v e r y i m p o r t a n t a n d has o n l y a s m a l l i n f l u e n c e o n t h e o r i e n t a t i o n and on the emission anistropy. (4) T h e o r i e n t a t i o n s o f B C h l c a n d B P h e c in the s a m e m a t r i x are similar, b u t the p r e s e n c e o f Mg influences the interactions between LC and pigment molecules, changing the distribution of p i g m e n t in L C .
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