5 Photoinduced electron transfer from Phycoerythrin to colloidal TiO2 and metal-TiO2 nanoparticles

5.1. Introduction

Solar energy conversion based on dye-sensitization of wide band gap

nanocrystalline semiconductor is an area of intense investigation [1–5].

The most efficient dye-sensitized solar cells (DSSC) to date are based on

ruthenium-containing metallorganic dyes adsorbed on nanocrystalline TiO2,

the best of which have been reported to convert solar energy to electrical

energy with an efficiency of 10–11% [6,7]. Photoexcitation of N3 dye

[cis-di-(thiocyanato)bis(4,4′-dicarboxy-2,2′-bipyridine) ruthenium(II)], results in

an intramolecular metal-to-ligand charge-transfer transition. The photoexcited

electrons located in the bipyridyl ligands can be efficiently injected into the

conduction band of TiO2 electrode on an ultrafast time scale via carboxyl groups

anchored to the TiO2 surface. Conversely, recombination between the electrons

injected into TiO2 and the cations of the N3 dye is a slow process [8], apparently

due to the large separation between TiO2 and Ru3+ imposed by the bipyridyl

ligands. Organic dyes have also been utilized as photosensitizers in DSSC.

Organic dyes have several advantages as photosensitizers: (a) they are cheaper

than Ru complexes, (b) they have large absorption coefficients due to

intramolecular δ-δ* transitions and (c) there are no concerns about limited

resources, because they do not contain noble metals such as ruthenium. DSSC

based on metal-free organic dyes [9-26], porphyrin dyes [27-32] and natural dyes

[33-35] have been studied and developed. Since the efficiencies of DSSC have not

yet approached the theoretical limit and are not competitive with the more

expensive silicon-based solar cells, their main advantage of cost-effectiveness

depends on the utilization of cheap and readily available sensitizer dyes. So, the

use of nontoxic natural pigments as sensitizers would definitely enhance the

Photoinduced electron … 106

environmental and economic benefits of this alternative form of solar energy

conversion.

The phycobiliproteins are antennae-protein pigments involved light

harvesting in cyanobacteria, rhodophytes, cryptomonads and cyanelles [36]. In

cyanobacteria and red algae, the phycobiliproteins are organized in

supramolecular complexes called phycobilisomes which are assembled in regular

arrays on the outer surface of the thylakoid membranes. Phycobiliproteins are

oligomeric and built up from chromophore bearing polypeptides belonging to the

α and β families of polypeptides [37]. The colors of phycobiliproteins originate

mainly from covalently bound prosthetic groups that are open-chain tetrapyrrole

chromophores namely phycobilins (possessing A, B, C and D rings). They are

either blue colored phycocyanobilin (PCB), red colored phycoerythrobilin (PEB),

yellow colored phycourobilin (PUB) and purple colored phycobiliviolin (PVB),

also named cryptoviolin. These chromophores are generally bound to the

polypeptide chain at conserved positions either by one cysteinyl thioether linkage

through the vinyl substituent on the pyrrole ring A of the tetrapyrrole or

occasionally by two cysteinyl thioether linkages through the vinyl substituent on

both A and D pyrrole rings [38]. Four main classes of phycobiliproteins are exist

in nature: Allophycocyanin (APC, bluish green), phycocyanin (PC, blue),

phycoerythrin (PE, purple) and phycoerythrocyanin (PEC, orange) having

absorption in the range of 650-655 nm, 615-640 nm, 565-575 nm and 575 nm

respectively and emit light at 660 nm, 637 nm, 577 nm and 607 nm respectively

[39]. Phycobiliproteins are used as colorants in food, cosmetic and pharmaceutical

industry [40], possess curative properties and used as fluorescence tags in

biomedical research [41,42].

The choice of phycoerythrin pigment as a sensitizer [Scheme 1] is due to

its significant visible light absorption and the presence of –COO− group which

Photoinduced electron … 107

could serve as an anchoring group between the pigment and TiO2 surface. The

spectral properties, such as (i) it contains multiple bilin chromophores and hence

high absorbance coefficients over a wide region of visible spectra

(ε = 2.5 x 106 M−1 cm−1 at 563 nm); (ii) high fluorescence quantum yield

(Φ = 0.98) independent of pH; (iii) strong absorption at 563 nm and strong

emission at 580 nm. It extend well into the red region of the visible spectrum,

where interference from biological molecules is minimal; (iv) large stokes shift

that minimizes interferences from Rayleigh and Raman scattering and other

fluorescing species; (v) highly soluble in aqueous solutions; and (vi) stable in

solution as well as solid phase, thus it can be stored for long periods. The

phycocyanin pigment have longer lifetime (nano seconds) than widely employed

N3 or N719 Ru based dyes (femto seconds) [43-45].

Photosensitization of wide-band gap semiconductors such as TiO2 by

visible light absorbing dyes has become more practical for solar cell applications

in the conversion of light into electricity [46]. Sensitization of colloidal TiO2 has

been studied extensively in the past [47-51]. Recently we have reported the

sensitization of colloidal TiO2 nanoparticles using porphyrins [52].

Semiconductor particles of colloidal dimensions are sufficiently small to yield

transparent solutions, allowing direct analysis of electron transfer by fluorescence

quenching technique [53].

N N N N

H H H H

O O

SHH3CH3C

HH

HOOC COO

__

H

Scheme 1: Structure of Phycoerythrin

Photoinduced electron … 108

Our interest is to investigate the process of electron transfer from excited

phycoerythrin to the conduction band of TiO2 by using absorption and

fluorescence spectroscopic measurements. From such studies, we can understand

the feasibility of flow of electrons from conduction band of TiO2 into the metal

core based on the energetic calculations. Electrons stored in metal core can be

readily discharged or scavenged on demand by electron acceptors as illustrated in

Scheme 2. Further, the mechanism for electron transfer process on the basis of

energy level diagram has also been proposed in this chapter. To the best of our

knowledge this is the first attempt of using phycoerythrin as a photosensitizer for

colloidal AuTiO2, AgTiO2 and TiO2 nanoparticles.

TiO2

VB

CB

e

Phycoerythrin*

νhe

e_

Au/Agacceptors

Scheme 2: Photoinduced charge injection and charge separation.

5.2. Experimental section 5.2.1. Materials Titanium (IV) 2-propoxide, Uniblue, Acid blue, Alizarin red S and Alizarin

were purchased from Aldrich. Phycoerythrin (PE) was obtained as gift sample

from Dr. S. Sekar, Bharathidasan University, Trichy. The doubly distilled water

was used for preparing the solutions. All measurements were performed at room

temperature.

Photoinduced electron … 109

5.2.2. Methods 5.2.2.1. Preparation of colloidal TiO2 nanoparticles

The colloidal TiO2 suspension was prepared by the hydrolysis of

titanium(1V) 2-propoxide [54] as described in chapter 4, section 4.2.2.1.

5.2.2.2. Preparation of colloidal AuTiO2 nanoparticles

Colloidal AuTiO2 nanoparticles in water were prepared by electrostatic

adsorption of AuCl4− ions on TiO2 surface followed by its reduction with NaBH4

[55] as described in chapter 4, section 4.2.2.2.

5.2.2.3. Preparation of colloidal AgTiO2 nanoparticles

The method of preparation of colloidal AgTiO2 nanoparticles in water was

similar to the one employed earlier [55] as described in chapter 4, section 4.2.2.3.

5.2.2.4. Preparation of phycoerythrin 5.2.2.4.1. Organism and culture conditions

Phycoerythrin employed in this study was obtained from the cyanobacteria

namely Anabaena sp. (fresh water form) from the culture collections maintained

in the Department of Biotechnology, Bharathidasan University, Tiruchirappalli.

Anabaena sp. was cultured in BG II fresh water medium [56] at 27±2° C with

artificial illumination from cool white fluorescent lamps.

5.2.2.4.2. Phycobiliprotein Extraction

Phycoerythrin was extracted from the freshly harvested biomass by the

method of freeze thawing in distilled water [57]. The biomass was frozen at

−20° C for 48 h and then thawed at room temperature with the addition of distilled

water (1 ml of water / gm of biomass). This is followed by centrifugation at

10,000 rpm for 10 min at 4° C.

Photoinduced electron … 110

5.2.2.4.3. Ammonium sulfate fractionation Finely powdered ammonium sulfate was gradually added into the crude

extract to obtain 35% saturation with continuous stirring for one hour. The

resulting solution was kept overnight in dark and the precipitation was collected

by centrifugation at 10,000 rpm for 10 min at 4° C. The pellets obtained from

ammonium sulfate precipitation was suspended in a small volume of distilled

water and subjected to dialysis for 10 h against 100 times volume of distilled

water [58].

5.2.2.5. Fluorescence quenching experiments

Samples were prepared by dissolving phycoerythrin (PE) in water and

administering the appropriate amounts of colloidal TiO2, AuTiO2 and AgTiO2

nanoparticles. The samples were deoxygenated by bubbling with pure nitrogen.

Quartz cells (4 x 1 x 1 cm) with high vacuum Teflon stopcocks were used for

bubbling.

5.2.2.6. Steady-state measurements

The steady-state fluorescence quenching measurements were carried out in

a JASCO FP-6500 spectrofluorimeter. Excitation and emission wavelengths of PE

are 563 nm and 580 nm respectively. The slit widths (each 5 nm) and scan rate

(500 nm/min) were maintained constant for all the measurements. Absorption

spectral measurements were recorded using Cary 300 UV-Visible

spectrophotometer.

5.2.2.7. Time-resolved measurements

Fluorescence lifetime measurements were carried out in a picosecond time

correlated single photon counting (TCSPC) spectrometer. The excitation source

was the tunable Ti-sapphire laser (TSUNAMI, Spectra Physics, USA). The diode

Photoinduced electron … 111

laser pumped Millenia V (Spectra Physics) CW Nd-YVO4 laser was used to

pump the sapphire rod in the Tsunami mode locked picosecond laser (Spectra

Physics). The diode laser output was used to pump the Nd-YVO4 rod in the

Millennia. The PE was excited by the laser pulse at 563 nm. The time resolved

fluorescence emission was monitored at 580 nm. The emitted photons were

detected by a MCP-PMT (Hamamtsu R3809U) after passing through the

monochromator (f/3). The laser source was operated at 4MHz and the signal from

the photodiode was used as a stop signal. The signal from the MCP-PMT was

used as start signal in order to avoid the dead time of the TAC. The difference

between the start and stop signal is due to the time taken by the pulses traveling

through the cables and electronic relaxation of the excited state. The data analysis

was carried out by the software provided by IBH (DAS-6). The kinetic trace was

analyzed by non-linear least square fitting of mono exponential function.

5.3. Results and Discussion 5.3.1. Absorption characteristics of PE with TiO2 and metal-TiO2

Figures 1a, 1b & 1c shows the absorption spectra of phycoerythrin (PE) in

the absence and presence of colloidal AuTiO2, AgTiO2 and TiO2 nanoparticles at

different concentrations. In the presence of colloidal AuTiO2 nanoparticles the

absorbance of PE at 563 nm was increased with the peak shift around 5 nm

[Figure 1a]. This implies that there is a surface interaction of phycoerythrin with

colloidal AuTiO2 through carboxyl group [Scheme 3], similar to the interaction of

fluorescein molecules with colloidal TiO2 reported by Marcus Hilgendorff and co-

workers [59]. The changes in intensity of the absorption peak at 563 nm indicate

the formation of surface complex. Similar type of spectral behaviour has been

observed for PE−AgTiO2 and PE−TiO2 systems [figures 1b & 1c].

Photoinduced electron … 112

0

0.4

0.8

1.2

1.6

450 500 550 600 650 700 750

Wavelength (nm)

Abs

orba

nce 0

2468

10

0 0.2 0.4 0.6 0.8 1

1/[AuTiO2] x 10-4 M

1/A

-A0

Figure 1a: Absorption spectrum of PE in the presence of colloidal AuTiO2 nanoparticles in the concentration range of 0−5 x 10−4 M in water. The inset is the straight line dependence of 1/Aobs−A0 on the reciprocal concentration of AuTiO2.

0

0.3

0.6

0.9

1.2

1.5

450 500 550 600 650 700 750

Wavelength (nm)

Abs

orba

nce 0

5

10

15

0 0.2 0.4 0.6 0.8 1

1/[AgTiO2] x 10-4 M

1/A

-A0

Figure 1b: Absorption spectrum of PE in the presence of colloidal AgTiO2 nanoparticles in the concentration range of 0−5 x 10−4 M in water. The inset is the straight line dependence of 1/Aobs−A0 on the reciprocal concentration of AgTiO2.

Photoinduced electron … 113

0

0.2

0.4

0.6

0.8

1

1.2

1.4

450 500 550 600 650 700 750

Wavelength (nm)

Abs

orba

nce 0

5

10

15

0 0.2 0.4 0.6 0.8 1

1/[TiO2] x 10-4 M

1/A

-A0

Figure 1c: Absorption spectrum of PE in the presence of colloidal TiO2 nanoparticles in the concentration range of 0−5 x 10−4 M in water. The inset is the straight line dependence of 1/Aobs−A0 on the reciprocal concentration of TiO2.

R-COO_

TiO2 R-COO_......TiO2+

R = PhycoerythrinAdsorption through

electrostatic interaction

Ag/AuAg/Au

Scheme 3: Electrostatic interaction of PE with positively charged colloidal TiO2 surface

The change in absorption spectra indicates that PE molecules adsorbed on

the surface of semiconductor nanoparticle to form a complex of the type

PE...M-TiO2.

PE + M-TiO2 PE.....M-TiO2

Kapp

→ (1)

[PE.....M-TiO2][M-TiO2][PE]Kapp =

Photoinduced electron … 114

The apparent association constant for the formation of this type of surface

complex (Kapp), can be estimated from the changes in absorbance at 563 nm by

using Benesi–Hildebrand equation (2) [60].

Aobs = (1-α)C0εPEl + αC0εcl → (2)

where Aobs is the observed absorbance of the PE solution containing different

concentrations of colloidal M-TiO2 at 563 nm; α is the degree of association

between PE and M-TiO2; εPE and εc are the molar extinction coefficients at the

defined wavelength for PE and the formed complex, respectively, ‘C0’ is the

initial concentration of free PE. Equation (2) can be expressed by equation (3),

where A0 and Ac are the absorbance of PE and the complex at 563 nm,

respectively, with the concentration of C0:

Aobs = (1−α)A0 + αAc → (3)

At relatively high M-TiO2 concentrations, α can be equated to

(Kapp[M-TiO2])/(1+Kapp[M-TiO2]). In this case, equation (3) can be expressed as

equation (4):

Aobs-A0 Ac-A0 Kapp(Aobs-A0)[M-TiO2]+=1 1 1

→ (4)

The inset of figures 1a-c shows the Benesi-Hildebrand plot and there is a good

linear dependence of 1/(Aobs-A0) on the reciprocal concentration of semiconductor

nanoparticles. The values of Kapp determined from the plots were shown in

Table 1. Further, the reason for higher association constant value for PE−AuTiO2

compared to AgTiO2 and TiO2 may be due to the larger surface area of AuTiO2.

When the surface area is decreased, the effective electron acceptor nature should

be decreased [61].

Photoinduced electron … 115

5.3.2. Fluorescence quenching characteristics

Figures 2a, 2b & 2c shows the effect of increasing concentration of

colloidal AuTiO2, AgTiO2 and TiO2 nanoparticles on the fluorescence emission

spectrum of phycoerythrin. Addition of colloidal AuTiO2 nanoparticles to the

solution of phycoerythrin resulted in the quenching of its fluorescence emission

[Figure 2a]. This quenching behaviour is similar to the earlier reported studies

[62]. Similar type of spectral behaviour has been noticed for PE−AgTiO2 and

PE−TiO2 [figure 2b & 2c].

The apparent association constant (Kapp) has been obtained from the

fluorescence quenching data according to the following equation (5),

F0-F' Kapp(F0-F')[M-TiO2]+=1 1 1F0-F → (5)

where Kapp is the apparent association constant, F0 is the initial fluorescence

intensity of phycoerythrin, F′ is the fluorescence intensity of phycoerythrin

adsorbed on colloidal M-TiO2 and F is the observed fluorescence intensity at its

maximum. The plot of 1/F0-F versus 1/[M-TiO2] is shown in the inset of

figures 2a-c representing a good linear relationship between 1/F0-F and the

reciprocal concentration of colloidal M-TiO2. From the slope, the value of Kapp

has been calculated and these values are shown in table 1.

Table 1: The apparent association constant (Kapp) for the PE-MTiO2 systems.

S.No. Systems aKapp (x 102 M−1) bKapp (x 102 M−1)

1 PE-AuTiO2 4.39 3.43

2 PE-AgTiO2 2.35 2.11

3 PE-TiO2 1.72 1.67

a obtained from absorption studies b obtained from fluorescence studies

Photoinduced electron … 116

0

100

200

300

400

500

600

560 570 580 590 600 610 620 630 640

Wavelength (nm)

Inte

nsity

0

0.005

0.01

0.015

0.02

0 0.2 0.4 0.6 0.8 1

1/[AuTiO2] x 10-4 M

1/F 0

-F

Figure 2a: Fluorescence quenching of PE in the presence of colloidal AuTiO2 nanoparticles in the concentration range of 0−5 x 10−4 M in water. The inset is the straight line dependence of 1/(F0-F) on the reciprocal concentration of AuTiO2.

0

100

200

300

400

500

600

560 570 580 590 600 610 620 630 640

Wavelength (nm)

Inte

nsity

00.005

0.010.015

0.020.025

0 0.2 0.4 0.6 0.8 1

1/[AgTiO2] x 10-4 M

1/F 0

-F

Figure 2b: Fluorescence quenching of PE in the presence of colloidal AgTiO2 nanoparticles in the concentration range of 0−5 x 10−4 M in water. The inset is the straight line dependence of 1/(F0-F) on the reciprocal concentration of AgTiO2.

Photoinduced electron … 117

0

100

200

300

400

500

600

560 570 580 590 600 610 620 630 640

Wavelength (nm)

Inte

nsity

0

0.01

0.02

0.03

0.04

0 0.2 0.4 0.6 0.8 1

1/[TiO2] x 10-4 M

1/F 0

-F

Figure 2c: Fluorescence quenching of PE in the presence of colloidal TiO2 nanoparticles in the concentration range of 0−5 x 10−4 M in water. The inset is the straight line dependence of 1/(F0-F) on the reciprocal concentration of TiO2. The Kapp value is decreased in the following order:

AuTiO2 > AgTiO2 > TiO2

Among the above three systems pure TiO2 shows lesser Kapp. While comparing

AuTiO2 and AgTiO2 the former one shows more quenching efficiency than the

latter due to the higher Fermi level of Au (0.75 V) than Ag (0.45 V).

The ability of the excited state phycoerythrin to inject its electrons into the

conduction band of TiO2 is determined from the energy difference between the

conduction band of TiO2 and excited state oxidation potential of phycoerythrin.

According to the equation Es*/s+ = Es/s+ − Es, the oxidation potential of excited

state phycoerythrin is calculated as −1.77 V vs SCE, where, Es/s+ is the oxidation

potential of phycoerythrin, 0.36 V vs SCE and Es is the excited state energy,

2.13 eV (Excited state energy of the phycoerythrin is calculated from the

fluorescence maximum based on the reported method [63]). The energy level of

the conduction band of TiO2 is −0.1 V vs SCE [64]. It suggests that the electron

Photoinduced electron … 118

transfer from excited state phycoerythrin to the conduction band of TiO2 is

energetically favorable [Scheme 4]. The electrons injected into the conduction

band of TiO2 are quickly transferred to the metal core and it leads to the

suppression of back electron transfer process [Scheme 2&4]. Hence, AuTiO2 is

more efficient in suppressing the back electron transfer process while compared to

AgTiO2 and pure TiO2 nanoparticles.

1

2

3

0

-1

-2

TiO2

CB

VB

(0.36 V)(-0.1 V)

V vs SCE

(2.9 V)

Es/s+

Es*/s+(-1.77 V)

νh

Phycoerythrin0.45 V

Ag 0.75 VAu

Scheme 4: Schematic diagram describing the electron-donating energy level of PE.

5.3.3. Fluorescence lifetime measurements As shown by previous reports [65-67], the dye molecules adsorbed on the

semiconductor surface had significantly shorter fluorescence lifetime than the

unadsorbed molecules. The fluorescence decay of PE (1 x 10−6 M) in the absence

and presence of metal-semiconductor nanoparticles are shown in figures 3 (a, b

and c for PE with AuTiO2, AgTiO2 and TiO2 respectively). In the absence of

metal-semiconductor nanoparticles, the decay curve of PE is fitted with single

Photoinduced electron … 119

exponential decay (F(t) = A exp (−t/τ)) with lifetime of 7.2 ns. Upon addition of

metal-semiconductor nanoparticles (5 x 10−4 M), the decay of PE is deviated from

single exponential decay to bi-exponential decay (F(t) = A1 exp (−t/τ1) + A2 exp

(−t/τ2)). It shows shorter-lifetime and longer-lifetime components. The shorter

lifetime is attributed to the adsorbed PE (τads) and the longer one is for

free/unadsorbed PE (τ). The fluorescence lifetime of PE with metal

semiconductor nanoparticles are given in Table 2. Further these lifetime data

clearly indicates that more electron transfer is possible in the case of Au-TiO2

system compared to pure TiO2 system because of the flow of electrons from the

conduction band of TiO2 into the metal core having low lying Fermi level which

is energetically favorable (Scheme 4).

1

10

100

1000

10000

0 5 10 15 20 25 30

Time (ns)

Cou

nts

Figure 3a: Fluorescence decay of PE in the absence and presence of colloidal AuTiO2 nanoparticles (5 x 10−4 M) in water.

Photoinduced electron … 120

1

10

100

1000

10000

0 5 10 15 20 25 30

Time (ns)

Cou

nts

Figure 3b: Fluorescence decay of PE in the absence and presence of colloidal AgTiO2 nanoparticles (5 x 10−4 M) in water.

1

10

100

1000

10000

0 5 10 15 20 25 30

Time (ns)

Cou

nts

Figure 3c: Fluorescence decay of PE in the absence and presence of colloidal TiO2 nanoparticles (5 x 10−4 M) in water.

Photoinduced electron … 121

The observed decrease in lifetime could be correlated with the electron transfer

process in the dye molecules adsorbed on the semiconductor nanoparticles by

using equation (6),

ket = 1/τads – 1/τ → (6)

where, τ and τads are the lifetime of free PE molecules in aqueous solution and

adsorbed on the semiconductors surface and ket is the specific rate of electron

transfer process. By substituting the values of τ and τads in the above equation (6)

the values of ket were calculated and shown in Table 2. The presence of noble

metals promotes the interfacial electron transfer process in the excited state of PE

to the metal-semiconductor nanoparticles [Scheme 2&4] and hence higher

ket was obtained for Au and AgTiO2 compared to pure TiO2.

Table 2: Fluorescence lifetime of PE (1 x 10−6 M) in presence of metal- semiconductors (5 x 10−4 M) and the rate of electron transfer (ket).

S. No. Sensitizer τ (ns) τads (ns) ket (x 109 s−1)

1 PE 7.2 - -

2 PE+TiO2 7.1 0.22 4.40

3 PE+AgTiO2 7.1 0.19 5.12

4 PE+AuTiO2 7.0 0.12 8.19

5.3.4. Calculation of free energy change (∆Get) for electron transfer reactions The bandgap energy of TiO2 (3.2 eV) is greater than the excited state

energy (2.13 eV) of phycoerythrin and there is no overlap between the

fluorescence emission of phycoerythrin (580 nm) with the absorption of colloidal

Photoinduced electron … 122

TiO2 (350 nm). Thus energy transfer from excited state phycoerythrin to colloidal

TiO2 has been ruled out.

Therefore it is concluded that the fluorescence quenching shown in

figure 2a, 2b & 2c is caused by electron transfer. The thermodynamic feasibility

of excited state electron transfer reaction was confirmed by the calculation of free

energy change by employing the well known Rehm-Weller expression [68].

∆Get = E½(ox)

− E½(red) − Es + C → (7)

where, E½(ox) is the oxidation potential of phycoerythrin (0.36 V), E½

(red) is the

reduction potential of TiO2 (i.e.) conduction band potential of TiO2, −0.1 V, Es is

the excited state energy of phycoerythrin and C is the coulombic term. Since one

of the species is neutral and the solvent used is polar in nature, the coulombic term

in the above expression can be neglected [69]. The value of ∆Get is calculated as

−1.67 eV and this higher negative value indicates that the electron transfer process

which occurred in this system is thermodynamically favorable [70,71].

5.3.5. Photoreduction of Anthraquinone dyes The electron transfer process has also been proved by the comparative

study of phycoerythrin with certain anthraquinone dyes. Anthraquinone dyes are

very good electron acceptors [Scheme 5] [72,73], so they have used to probe the

electron transfer process in phycoerythrin. Figures 4a, 4b, 4c & 4d shows the

fluorescence spectrum of PE in the absence and presence of anthraquinone dyes in

different concentration range. From the fluorescence study we observed that the

emission intensity of PE gradually decreases with increasing the dye

concentration, this clearly indicates the quenching of PE was occurred.

The fluorescence quenching of PE can be explain based on the well known

Stern-volmer relationship as shown in equation (8).

I0/I = 1 + Ksv [Q] → (8)

where I0 and I are the intensity of PE in the absence and the presence of dye,

Photoinduced electron … 123

Ksv is Stern-Volmer constant and [Q] is the concentration of the dye.

The bimolecular quenching rate constant (kq) was calculated [Table 3] using

equation (9).

Ksv = τ . kq → (9)

where, τ is the fluorescence lifetime of PE in the absence of quencher, kq is

the bimolecular quenching rate constant. The plot between I0/I vs [Q] were linear

for all dyes, indicating dynamic nature of quenching process [Figure 5].

HN

NH2

SO3Na

SOO

CH2

Uniblue

O

O

HN

NH2

SO3Na

Acid blue

O

O

Alizarin Red SO

O

OH

OH

SO3Na

AlizarinO

O

OH

OH

Scheme 5: Structure of Anthraquinone dyes

Photoinduced electron … 124

0

100

200

300

400

500

600

560 570 580 590 600 610 620 630 640

Wavelength (nm)

Inte

nsity

Figure 4a: Fluorescence quenching of PE in the presence of Uniblue in the concentration range of 0−5 x 10−4 M in water.

0

100

200

300

400

500

600

560 570 580 590 600 610 620 630 640

Wavelength (nm)

Inte

nsity

Figure 4b: Fluorescence quenching of PE in the presence of Acid blue in the concentration range of 0−5 x 10−4 M in water.

Photoinduced electron … 125

0

100

200

300

400

500

600

560 570 580 590 600 610 620 630 640

Wavelength (nm)

Inte

nsity

Figure 4c: Fluorescence quenching of PE in the presence of Alizarin Red S in the concentration range of 0−5 x 10−4 M in water.

0

100

200

300

400

500

600

560 570 580 590 600 610 620 630 640

Wavelength (nm)

Inte

nsity

Figure 4d: Fluorescence quenching of PE in the presence of Alizarin in the concentration range of 0−5 x 10−4 M in water.

Photoinduced electron … 126

0.8

1

1.2

1.4

1.6

1.8

2

0 1 2 3 4 5

[Q] x 10-4 M

I0/I

Uniblue Acid blue Alizarin Red S Alizarin

Figure 5: Stern-Volmer plot for the fluorescence quenching of PE with various concentrations of dyes (0 − 5 x 10−4 M) in Water.

The quenching rate constant decreased in the following order:

Uniblue > Acid blue > Alizarin Red S > Alizarin

Among the dyes, Uniblue possess highest quenching rate constant than

acid blue. This is due to the fact that uniblue has one additional vinyl sulfone

group which is an electron withdrawing group that makes the uniblue more

electron deficient compared to acid blue and hence enhancing it’s ability to

quench the PE.

Alizarin Red S has higher quenching rate constant than Alizarin. These

two dyes are almost structurally similar, but in the case of Alizarin Red S, one

sulfonate group is attached in the anthraquinone system, so the electron density in

the quinone group is reduced, making it relatively more electron deficient and

hence enhancing its ability to quench the PE via electron transfer.

Photoinduced electron … 127

The nature of electron transfer pathway (i.e., oxidative or reductive

quenching of the PE excited state) can be understood by examining the free

energy of the corresponding electron transfer reactions. Thermodynamics of

electron transfer from PE to the quencher can be calculated by the well known

Rehm-Weller equation (7).

The calculated ∆Get values are given in Table 3. The negative values of

free energy change indicated the electron transfer from PE to dyes is

thermodynamically favorable [Scheme 6].

PE

PE* + Q (PE.....Q)* (PE +.....Q )* PE + + Q

PE + Q Products

h

k d

kd

k et

ket kesc

kRkb

_ _

ν

__

Scheme 6: Mechanism of fluorescence quenching of PE by dyes

where, kd and k−d are the rate constants of diffusion and dissociation of encounter

complex, respectively. ket and k−et are the activation controlled rate constants of

electron transfer, and kesc is the rate constant for the separation of radicals. kb is

the rate constant for the recombination of radical pair. kR is the rate constant for

decay of PE radical.

Photoinduced electron … 128

Table 3: Fluorescence quenching rate constants and thermodynamic data of PE by anthraquinone dyes.

S. No. Quencher kq (x 1010 M–1s–1)a E1/2 vs SCE (V)b ∆Get (eV)c

1 Uniblue 20.6 0.65 -2.42

2 Acid blue 15.9 0.66 -2.43

3 Alizarin red S 12.6 0.67 -2.32

4 Alizarin 8.7 0.55 -2.44

a determined by steady state fluorescence quenching in water b reduction potential of dyes in V vs SCE in water. c calculated by Rehm-Weller equation.

5.4. Conclusions The interaction of phycoerythrin with colloidal metal-semiconductor

nanoparticles has been studied by absorption, steady state and time resolved

fluorescence spectroscopic methods. Phycoerythrin adsorbed on the surface of

metal semiconductor nanoparticles through its carboxyl group, as evidenced by

the effect of colloidal metal semiconductor nanoparticles concentration on the

absorption study. The apparent association constants were calculated from both

the absorption and fluorescence changes and they were agreed well. Based on the

energy level diagram and more negative free energetics, it is suggested that the

metal core has effect on the electron transfer from excited state phycoerythrin to

the conduction band of TiO2. On the other hand, presence of metal core

suppresses the charge recombination process. Electron accumulation within the

metal core is likely to influence the overall charge separation in the composite

system. The electron transfer process has also been proved by the comparative

study of phycoerythrin with certain anthraquinone dyes. Insinuation of the metal

semiconductor nanoparticles is improving the performance of dye-sensitized solar

cells.

Photoinduced electron … 129

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