Synthesis of new ESIPT-fluorescein: Photophysics of pH sensitivity and fluorescence

Article abstract of DOI:10.1021/jp2073123

ESIPT-inspired benzimidazolyl substituted fluorescein dyes were synthesized. PH-sensitivity was determined by the photophysical property measured at a physiological possible pH range. Fluorescence quantum efficiency values were calculated independently at two different emissions. A rational relationship is defined between fluorescence quantum efficiency and calculated HOMO energy.

Full text of DOI:10.1021/jp2073123

ARTICLE
pubs.acs.org/JPCA
Synthesis of New ESIPT-Fluorescein: Photophysics of pH Sensitivity
and Fluorescence
Vikas S. Patil, Vikas S. Padalkar, Kiran R. Phatangare, Vinod D. Gupta, Prashant G. Umape, and
Nagaiyan Sekar*
Institute of Chemical Technology (Formerly UDCT), N. P. Marg, Matunga, Mumbai, 400 019 Maharashtra, India
S Supporting Information
b
ABSTRACT: ESIPT-inspired benzimidazolyl substituted fluo-
rescein dyes were synthesized. PH-sensitivity was determined by
the photophysical property measured at a physiological possible
pH range. Fluorescence quantum efficiency values were calculated
independently at two different emissions. A rational relationship is
defined between fluorescence quantum efficiency and calculated
HOMO energy.
’
INTRODUCTION
may be enhanced or diminished relative to the emission at
another. Ratios between these signals then can be calibrated to
indicate pH values. Advantages when using ratiometric methods
are accrued because parameters such as optical path length, local
probe concentration, photobleaching, and leakage from the cells
are irrelevant. This must be so since both signals come from the
probe in exactly the same environment.
Fluorescence probes are excellent sensors for biological pH for
the reasons that they are sensitive combined with fast response.
Fluorescent pH sensors find wide use in analytical chemistry,
bioanalytical chemistry, cellular biology (for measuring intracel-
1
ꢀ3
lular pH), and medicine.
Among other pH measurement
methods, fluorescence spectroscopy has advantages with respect
to spatial and temporal observation of pH changes. They tend to
be operationally simple, and they are in most cases nondestruc-
tive to cells. Development of optical pH chemosensors,
specific requirement of highly sensitive indicators within the
physiological pH range, is currently a fertile area of research.
In this area, fluorescein and its derivatives are perhaps the most
widely used fluorescent pH probes
photophysical properties.
However, fluorescence measurements are largely influenced
by many factors, including optical path length, changes of tem-
perature, altered excitation intensities, and varied emission col-
lection efficiencies. In order to circumvent these problems an
alternative, viz., a ratiometric detection technique is used.
Ratiometric fluorescent measurements make use of changes in
Fluorescent molecules exhibiting dual emission are those exhi-
biting excited state intramolecular proton transfer (ESIPT). A
variety of molecules have intramolecular hydrogen bonds (H-bonds)
that may be photo-induced to undergo proton transfer. ESIPT
can be observed in a variety of such molecules that contain both
4
ꢀ8
with
9
,10
18ꢀ26
hydrogen donor and acceptor in close proximity.
An intra-
11ꢀ14
molecular hydrogen bond generally formed in the ground state
due to their excellent
willmigrate to theneighboringprotonacceptor leadingtoa photo-
27ꢀ29
0
In the general family of 2-(2 -hydroxyphenyl)
3
tautomer.
benzimidazole, benzoxazole, benzthiazole, and benztriazole,
0ꢀ33
the ESIPT phototautomer gives rise to an emission with large
Stokes shift. The high intensity of fluorescence emission and
large Stokes shift due to intramolecular proton transfer pheno-
34ꢀ48
mena
allow these molecules to have many interesting appli-
15
cations. Molecules undergoing ESIPT exhibit dual fluorescence,
one a normal Stokes shifted fluorescence band and another origi-
nating from a tautomer formed in the exited state. Further the
fluorescence band maximum and the fluorescence quantum yield
the ratio of the intensities of the emission at two wavelengths.
Thus, ratiometric fluorescent sensors have an advantage that they
can be used to evaluate the analyte concentration with a provi-
sion of built-in correction for environmental effects. Ratiometric
fluorescence spectroscopic methods rely heavily on fluorescent
sensors that are differentially sensitive to protons for at least two
Received: August 1, 2011
16,17
excitation or emission wavelengths.
For example, for a suit-
Revised:
November 19, 2011
able fluorescent dye, emission at one carefully chosen wavelength
Published: December 05, 2011
r 2011 American Chemical Society
536
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|J. Phys. Chem. A 2012, 116, 536–545

The Journal of Physical Chemistry A
ARTICLE
Scheme 1. Synthesis of Fluorescein Derivatives (1aꢀ1e)
of the tautomer fluorescence band are very sensitive, whereas
those of the normal band do not depend much on the environ-
ment. It is commonly understood that the molecular struc-
ture of the ESIPT compounds regulates their abnormal photo-
physical properties to a considerable extent, in most cases
exceeding the influence of the environment on them. There is
an example 2-(3,5,6-trifluoro-2-hydroxy-4-methoxyphenyl)
benzoxazole, a fluorophore, that undergoes intramolecular
(Varian, Australia). Simultaneous DSC-TGA measurements
were performed on simultaneous DSC-TGA Waters (India)
Pvt. Ltd.
’
SYNTHESIS AND CHARACTERIZATION
Synthesis of 4-(1H-Benzo[d]imidazol-2-yl) Benzene-1,3-
diol (3). 2,4-Dihydroxy benzoic acid 1 (10 g, 64.9 mmol), and
o-phenylenediamine 2 (7.012 g, 64.9 mmol) were mixed in poly-
phosphoric acid (121.94 g). The mixture was stirred at 250 °C for
4 h, and then it was allowed to cool at room temperature and
poured into 1200 mL of ice-cold water with constant stirring.
A dark brown precipitate was obtained, which was filtered and
4
9
hydrogen bonding.
The molecules showing ESIPT particularly o-hydroxy benza-
zoles have absorption in the UV region and emission in the visi-
ble region. An attempt has been made in this work to incorporate
an ESIPT unit in a molecule having absorption in the visible
region.
dissolved in a cold solution of 10% Na CO (15.75 g dissolved in
2 3
1
37 mL of water) acidified with 1:1 HCl (40 mL) at 10 °C. This
solution was kept overnight at 0 °C to give the final product.
’
EXPERIMENTAL SECTION
1
Yield: 55%. Melting point = >300 °C. Mass: m/z 226 (M+). H
Materials and Equipment. All commercial reagents and
NMR ((CD ) SO, 300 MHz): δ 6.88 (d, 1H, 6.4 Hz), 6.88 (d,
3
2
solvents were procured from S.D. Fine Chemicals (India) and
were used without purification. The reaction was monitored by
TLC using 0.25 mm E-Merck silica gel 60 F254 precoated plates,
which were visualized with UV light. Melting points were mea-
sured on a standard melting point apparatus from Sunder
Industrial Product, Mumbai, and are uncorrected. The FT-IR
spectra were recorded on a Perkins-Elmer 257 spectrometer
using KBr discs. H NMR and C NMR spectra were recorded
on a VXR 300-MHz instrument using TMS as an internal
standard. Mass spectra were recorded on Lcq Advantage Max
Thermo Electron Corporation by negative mode analysis. The
visible absorption spectra of the compounds were recorded
on a Spectronic Genesys 2 UVꢀvisible spectrophotometer
and UVꢀvisible emission spectra were recorded on JASCOꢀ
FP 1520 and Cary Eclipse fluorescence spectrophotometer
1H, 6.4 Hz), 7.05 (s, 1H), 7.34 (s, 2H), 7.71 (s, 2H), 8.07 (s, 1H),
ꢀ1
11.2 (s, 1H) ppm. FT-IR (KBr, cm ): 3438 (phenolic OꢀH),
3322 (NꢀH), 1651 (imine CdN), 1571 (aromatic CdC), 1516,
1421, 1389, 1203 (phenol CꢀO).
General Procedure for the Synthesis of Fluorescein Deriv-
atives (1aꢀ1e). 4-(1H-Benzo[d]imidazol-2-yl) benzene-1,3-
diol 3 (88 mmol) was mixed with different anhydrides (4ꢀ8)
(44 mmol) in H SO and heated at 160 °C for 4 h and was al-
1
13
2
4
lowed to cool at room temperature. The reaction mixture was
poured into 100 mL ice-cold water and stirred for 15 min. The preci-
pitated product was filtered and washed with cold water (25 mL).
Purification of Fluorescein Derivatives (1aꢀ1e). The crude
product was purified by dissolving it into 10% Na CO solution
2
3
(46 mL) and acidified with 1:1 HCl (13 mL) to give the final
compounds (1aꢀ1e).
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The Journal of Physical Chemistry A
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Figure 1. Absorption spectra of 1a at various pH (6ꢀ13).
0
0
0
2
,7 -Di(1H-benzo[d]imidazol-2-yl)-3 ,6 -dihydroxy-3H-spiro-
0
7.76 (d, 2H, 10.5 Hz), 7.99 (s, 2H), 8.01 (s, 2H), 8.33 (s, 2H),
13
[
>
1
(
1
1
isobenzofuran-1-9 -xanthen]-3-one (1a). Yield: 55%. Mp =
11.2 (s, 2H) ppm. C NMR ((CD ) SO, 75 MHz): δ 101.98,
3
2
ꢀ
1
300 °C. FT-IR (KBr, cm ): 3432 (OꢀH), 3324 (NꢀH),
103.33, 110.97, 113.13, 123.61, 125.61, 129.02, 130.33, 131.02
145.78, 147.37, 159.47, 159.57. Elemental analysis: exptl, C =
70.25, H = 3.52, N = 10.80, O = 15.52; theor, C = 70.03, H = 3.59,
N = 10.89, O = 15.55
768 (lactone CdO), 1740 (lactone CdO), 1689 (CdO), 1642
CdN), 1578 (aromatic CdC), 1550 (aromatic), 1515, 1441,
402, 1339 (CꢀN), 1252 (phenol CꢀO), 1190 (lactone CꢀO),
1
0
0
0
0
130 (ether linkage). Mass m/z 564 (M+). H NMR ((CD ) -
2 ,7 -Di(1H-benzo[d]imidazol-2-yl)-3 ,6 -dihydroxy-3-oxo-
0
3
2
SO, 300 MHz): δ 6.58 (s, 2H), 7.17 (d, 2H, 6.4 Hz), 7.34(d, 2H,
.4 Hz), 7.68 (d, 2H, 6.4 Hz), 7.72 (dd, 2H, 6.4 Hz, 14.9 Hz),
3H-spiro[isobenzofuran-1-9 -xanthen]-5-carboxylic acid (1d).
ꢀ1
6
8
Yield: 57%. Mp = > 300 °C. FT-IR (KBr, cm ): 3277 (phenolic
OꢀH), 3210, 1759 (lactone CꢀO), 1731 (CdO), 1700 (CdO),
1667 (CdN), 1627, 1588 (aromatic CdC), 1496, 1404, 1337
(CꢀN), 1317, 1261, 1180 (lactone CꢀO), 1124 (lactone CꢀO),
.00 (s, 2H), 8.2ꢀ8.3 (d, 4H, 8.9 Hz), 8.4 (s, 2H), 11.3 (bs, 2H)
1
3
ppm. C NMR ((CD ) SO, 75 MHz): δ 85.46, 166.00, 106.29,
3
2
1
1
3
11.92, 115.21, 115.39, 124.11, 125.83, 127.29, 130.74, 141.71,
51.73, 152.48, 152.93. Elemental analysis: exptl, C = 72.25, H =
.52, N = 9.88, O = 15.52; theor, C = 72.33, H = 3.57, N = 9.92,
1
1060, 841, 805, 755, 668. Mass m/z 608 (M+). H NMR
((CD ) SO, 300 MHz): δ 6.00 (s, 1H), 7.15 (s, 2H), 7.52 (s, 1H),
3
2
O = 14.17.
7.55 (s, 1H), 7.66 (m, 8H, 10.0 Hz, 8.8 Hz), 7.02 (m, 2H, 11.4 Hz,
8.8 Hz), 7.84 (d, 1H, 11.4 Hz), 7.94 (d, 2H, 8.8 Hz), 8.34 (s, 1H),
0
0
0
0
2
,7 -Di(1H-benzo[d]imidazol-2-yl)-3 ,6 -dihydroxy-3H-spiro-
0
13
[
furan-2,9 -xanthen]-5(4H)-one (1b). Yield: 48%. Mp = >300 °C.
8.60 (s, 1H) ppm. C NMR ((CD ) SO, 75 MHz): δ 80.99,
3
2
ꢀ
1
FT-IR (KBr, cm ): 3312 (phenolic OꢀH), 2963(aliphatic CꢀH),
104.24, 108.35, 111.16, 111.46, 113.35, 123.45, 126.31, 129.98,
131.51, 132.19, 133.17, 145.50, 147.32, 153.50, 156.08, 159.40,
166.22, 168.13. Elemental analysis: exptl, C = 69.15, H = 3.32, N =
9.18, O = 18.32; theor, C = 69.08, H = 3.31, N = 9.21, O = 18.40.
2
1
924 (aliphatic CꢀH), 1768 (lactone CdO), 1689 (CdO),
642 (CdN), 1578 (aromatic CdC), 1515, 1441, 1402, 1339
(
CꢀN), 1252 (phenol CꢀO), 1190 (lactone CꢀO), 1005. Mass
1
0
0
0
0
m/z 514 (M+). H NMR ((CD ) SO, 300 MHz): δ 2.47 (t, 2H),
4-Amino-2 ,7 -di(1H-benzo[d]imidazole-2-yl)-3 ,6 -dihydroxy-
0
3 2
3
7
1
7
1
.57 (t, 2H), 7.71 (d, 2H, 9.2 Hz), 7.74 (dd, 2H, 9.2 Hz, 14.6 Hz),
.78 (s, 2H), 7.99 (s, 2H), 8.02 (s, 2H), 8.33 (m, 2H, 9.2 Hz,
7.4 Hz), 8.37 (m, 2H, 9.2 Hz) ppm. C NMR ((CD ) SO,
5 MHz): δ 40.32, 38.66, 101.98, 103.33, 110.97, 113.13, 123.61,
29.02, 130.33, 131.02 145.78, 147.37, 159.47. Elemental analysis:
3H-spiro-[isobenzofuran-1-9 -xanthen]-3-one (1e). Yield:
ꢀ
1
51%. Mp = >300 °C. Mass m/z 579 (M+). FT-IR (KBr, cm ):
3445, 1734 (ester CdO), 1700 (CdO), 1650 (CdO), 1558
(aromatic CdC), 1541, 1490, 1457, 1398, 1342 (CꢀN), 1258
(phenol CꢀO), 1177 (ester CꢀO), 1104 (ester CꢀO), 1048,
1
3
3
2
1
exptl, C = 69.75, H = 3.82, N = 10.78, O = 15.48; theorl, C =
869, 822, 668. H NMR ((CD ) SO, 300 MHz): δ 5.99 (s, 2H),
3
2
6
9.76, H = 3.90, N = 10.85, O = 15.49.
6.58 (s, 2H), 6.97 (s, 2H), 7.12 (s, 2H), 7.75 (m, 8H, 11.9 Hz,
14.2 Hz), 8.02 (m, 3H, 12.4 Hz, 11.9 Hz), 8.34 (s, 2H). C NMR
0
0
0
0
13
2
,7 -Di(1H-benzo[d]imidazol-2-yl)-3 ,6 -dihydroxy-3H-spiro-
0
[
furan-2,9 -xanthen]-5-one (1c). Yield: 41%. Mp =240ꢀ242 °C.
((CD ) SO, 75 MHz): δ 99.22, 101.52, 109.27, 111.37, 121.89,
3
2
ꢀ
1
FT-IR (KBr, cm ): 3310 (phenolic OꢀH), 2851 (aliphatic
CꢀH), 1734 (lactone CdO), 1696 (CdO), 1648 (CdN), 1598
123.44, 127.21, 128.61, 129.24, 145.58, 157.62, 157.75. Elemental
analysis: exptl, C = 70.38, H = 3.62, N = 12.01, O = 13.72; theor,
C = 70.46, H = 3.65, N = 12.08, O = 13.80.
Preparation of Working Solutions for Photophysical Mea-
surements. All the solutions were freshly prepared in water (pH
was maintained by 10% NaOH and 1:1 HCl) before recording
(
aromatic CdC), 1501, 1431, 1406, 1342 (CꢀN), 1252 (phenol
CꢀO), 1192 (lactone CꢀO), 1018. Mass m/z 516 (M+).
1
H NMR ((CD ) SO, 300 MHz): δ 6.55 (s, 2H), 7.69 (d, 2H,
3
2
8
.3 Hz), 7.71 (dd, 2H, 8.3 and 10.5 Hz), 7.74 (d, 2H, 10.5 Hz),
5
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Figure 2. Fluorescence spectra of 1a at various pH (6ꢀ13).
Figure 4. Fluorescence intensity against pH (compounds 1d and 1e).
Figure 3. Fluorescence intensity against pH (compounds 1a, 1b, and 1c).
Figure 5. Ratiometry of dual emission vs pH.
the absorption and emission spectra. The concentration of pre-
ꢀ
6
ꢀ1
(1aꢀ1e) have a basic fluorophoric unit (that is fluorescein molecule)
interacting with the ESIPT residue.
pared samples was 1 ꢁ 10 mol L , and the temperature dur-
ing analysis was at room temperature, 28 °C. For quantum yield
calculations, the sample concentration was 2 ppm to 10 ppm in
the same solvent.
The UVꢀvis absorption and fluorescence emission spectra
ꢀ
6
ꢀ1
of dyes (1aꢀ1e) at 1 ꢁ 10 mol L solution in water were
measured at different pH ranging from 6 to 13. Absorption and
emission maxima are also reported in the Table 1. UVꢀvisible
absorption-emission spectra of these dyes with respect to pH are
highly dependable.
’
RESULTS AND DISCUSSION
Synthesis and Characterization of Dyes. The reaction of
,4-dihydroxy benzoic acid 1 with o-phenylenediamine 2 in
Author: Presence of the ESIPT benzimidazole substituent
in the final structure of the dye molecule was found to be more
responsible for influencing the photophysical properties. In
addition to the ESIPT, the distribution of electron density on
the phthalein ring plays a major role in the photophysical be-
havior of dye molecules. When there is an electron withdrawing
group present on the phenyl ring (which does not contain a benz-
imidazolyle residue) of the xanthene chromophore, long wave-
length emission is observed. On the contrary, when electron re-
leasing substituent is present, a blue shift is observed. Dye 1a
has an excitation at 300 nm, which was totally unaffected by the
pH. It was observed that in the case of dye 1a in addition to the
usual excitation at 495 nm (characteristic of fluorescein molecule),
additional excitation occurred at 543 nm. It is interesting to
2
polyphosphoric acid at 250 °C furnished the required ESIPT
moiety 4-(1H-benzo[d]imidazol-2-yl)benzene-1,3-diol 3 with
good yield (Scheme 1, Supporting Information). The reaction
of 4-(1H-benzo[d]imidazol-2-yl)benzene-1,3-diol 3 with phthalic
anhydride 4, succinic anhydride 5, maleic anhydride 6, phthalic
anhydride-4-carboxylic acid 7, and 3-amino phthalic anhydride 8
in H SO at 160 °C for 4 h furnished the expected fluorescein
2
4
derived dyes 1aꢀ1e respectively (Scheme 1).
Photophysical Properties. The fluorescein derivatives
(
1aꢀ1e) contain two benzimidazole units, each adjacent to the
two hydroxyl groups of the fluorescein nucleus causing an ESIPT.
Unlike the known ESIPT fluorophores, these compounds
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Figure 6. Molecular orbital diagram and electron transfer mechanism in the excited state.
Figure 7. Structure and fluorescence quantum efficiency at different wavelength of fluorescein derivatives.
observe that this red-shifted excitation is not a function of pH
Table 1, Figure 1). Compound 1a contains three phenyl rings
300 nm has emission in the region 412ꢀ440 nm (Table 1;
Figures 3 and 4, Supporting Information). The additional con-
jugation is totally absent in the dye 1c, and it has an excitation
in the region 294ꢀ318 nm with a blue-shifted emission band
from 426 to 462 nm (Table 1; Figures 5 and 6, Supporting
Information). In the case of dye 1d, which carries an electron
withdrawing carboxyl substituent at the para position, the usual
excitation at 495 nm was found to be absent at pH 12 and 13.
However, an additional excitation at 545 nm was located
(Table 1; Figure 7, Supporting Information). Dye 1d showed a
strong emission band at the longer wavelength 572 nm and
comparatively weak emission band at 435 nm on excitation at
near 300 nm (Table 1; Figure 8, Supporting Information).
(
having conjugation. Dual emissions of dye 1a arising out of
excitation at 303 nm are located in the region 408ꢀ432 nm along
with a long wavelength emission at 570 nm (Table 1, Figure 2).
In the case of 1b, there are only two phenyl rings on the central
carbon atom and weakly conjugated two carbon atoms as against
the fully conjugated additional aromatic ring in 1a. Both of the
phenyl rings carry a benzimidazole moiety. Dual emission pheno-
mena are not found to be promising for the dyes 1b and 1c. In the
case of 1b, there is no additional conjugated phenyl ring to
central carbon atom of phthalein ring, and it has weakly conju-
gated two carbon atoms. The compound 1b on excitation at
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Figure 8. Fluorescein derivative divided into two parts: one electron donor phthalein moiety and electron acceptor xanthene (2,7-bis(1H-
benzimidazol-2-yl)-6-hydroxy-3H-xanthen-3-one) moiety.
Dye 1e contains an electron releasing amino substituent. For dye
1
e, an excitation band at 300 nm was observed along with a weak
excitation band located at 540 nm (Table 1; Figure 9, Supporting
Information). Dye 1e showed emission in the region 411ꢀ
4
43 nm on excitation at 300 nm (Table 1; Figure 10, Supporting
Information). Dual emission phenomenon was not found in 1e.
Effect of pH on Photophysical Properties. At pH 6, dye 1a
has three absorptions located at 303, 495, and 543 nm. The
absorption at 432 nm experiences a red shift beyond pH 6. At pH
values 7, 8, and 9, this red-shifted absorption band appears at
4
95 nm, while the other two absorptions at 303 nm and 543 nm
remain unchanged over the entire pH range. Upon raising the pH
above 9, the middle absorption band, which experienced a red
shift from 432 to 495 nm, disappeared completely (Table 1,
Figure 1). At all pH, the excitation at 303 nm was accompanied
by dual emission (Table 1, Figure 2), while another two absorp-
tions did not show any accompanying emissions. The short wave-
length emission located at around 408 nm (at pH 6) experienced
a red shift up to pH 9; the red-shifted emission appeared at
4
34 nm. Beyond pH 9, the emission maxima returned to the
original value around 410 nm. The long wavelength emission
remained constant through the entire pH range and was located
at around 570 nm (Table 1, Figure 2). The dye 1b, which is
devoid of a phenyl group at the central carbon atom, had a
different behavior. At pH values up to 9 only one absorption
centering at 300 nm was observed. Beyond pH 9, additional
absorption peaks appeared (Table 1; Figure 3, Supporting In-
formation). The excitation at 300 nm was accompanied by a
single emission at 436 nm (at pH 6), and it experienced a red shift
until the pH value was 9; the blue-shifted emission appeared at
Figure 9. HOMO energy of 2,7-bis(1H-benzimidazol-2-yl)-6-hydroxy-
H-xanthen-3-one as the electron acceptor (fluorophore) and the
phthalein moiety (electron donor).
3
For pH 8, at 490 nm an excitation band along with 535 nm was
located). The dye 1e showed photophysical properties similar to
1c except the fact that it showed dual emission at pH 11 (Table 1;
Figures 9 and 10, Supporting Information). Fluorescein has a
single emission at 520 nm. Incorporation of ESIPT in fluorescein
causes dual emission. ESIPT molecules exist in two forms (keto
and enol) at the first excited state. The existence of two forms
causes dual emission in compounds 1a and 1d; short wavelength
emission appears to be due to the enol form, and long wavelength
emission appears to be due to the keto form. After incorporation of
the ESIPT hydroxy benzimidazole moiety in fluorescein, the longer
emission experiences a red shift, and the emission occurs at 570 nm.
4
30ꢀ440 nm. From pH 10 onward, the same excitation gave dual
emission as shown in Table 1. The dual emission ceased to exist
at higher alkalinity, i.e., at pH 12 and pH 13 (Table 1; Figure 4,
Supporting Information). In the case of dye 1c where the central
carbon atom is conjugated to an aliphatic residue, there was no
dual emission at any pH value. The short wavelength excitation at
around 300 nm had a single emission located at 460 nm, which
experienced a blue shift from pH 11 onward (Table 1; Figures 5
and 6, Supporting Information). The dye 1d behaved in a similar
way to 1a except the fact that dual emission arising out of short
wavelength excitation was not sensitive to pH (Table 1; Figure 7,
Supporting Information). The short wavelength excitation was
located at 300 nm. An absorption located at 495 nm up to pH 11
disappeared beyond pH 11. Long wavelength absorption started
appearing at 545 nm from pH 9 onward (Table 1; Figure 8,
Supporting Information). For 1e, an excitation band near 300
was located for all pH values (except for pH 12 was located at
0
0
2-(2 ,4 -Dihydroxy phenyl) benzimidazole 3 absorbs at 326 nm
and has dual emission at 374 and 444 nm (Figure 11, Supporting
Information) After incorporation in to the fluorescein system,
1a shows dual emission at 434 and 570 nm and 1d at 435 and
572 nm. 1b, 1c, and 1e shows single emission at 440, 462, and
430 nm, respectively; these results clearly indicate that the
fluorescein moiety is responsible for the red shift of the longer
wavelength emission of 3.
2
85 nm), and a long wavelength excitation band for all pH values
The change in emission intensity with respect to pH values for
each emission wavelength irrespective of its emission band are
were located at 540 nm (except pH 12 was located at 490 nm, and
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Table 2. Physical Properties and Relative Fluorescence Quantum Yields of Compounds 1aꢀ1e
thermal stability
quantum yield at
quantum yield at
compd
mol wt
(°C (%))
short wavelength
long wavelength
total quantum yield
1
1
1
1
1
a
b
c
564.54
514.48
516.50
608.55
579.56
323(81.17)
300(81.38)
244(77.81)
335(72.84)
314(78.40)
ϕ1a 440 = 0.0879
ϕ1b 470 = 0.224
ϕ1c 448 = 0.117
ϕ1d 464 = 0.172
ϕ1e 436 = 0.3057
ϕ
1a 570 = 0.624
1d 567 = 0.79
0.7119
0.224
0.117
0.962
0.3057
d
e
ϕ
50,51
higher,
cence quantum efficiency at long wavelength emission was found
to be higherfor carboxylicacidsubstitutedfluorescein 1d (ϕ1d567
and the energy of HOMO was ꢀ9.12 eV. Fluores-
=
0
.79); increased reduction potential of the phthalein moiety was
50,51
observed,
and the energy of HOMO was ꢀ10.90 eV.
The photophysical properties of fluorescein derivatives are
usually understood by differentiating the molecule into two parts:
xanthene and phthalein (Figure 8). Energy values of HOMO are
independently represented in Figure 9. Our observation suggests
that the HOMO energy level of the phthalein moiety decides
whether electron transfer to the excited xanthene ring occurs. It
can be seen that low HOMO energy of the phthalein moiety
gives rise to high fluorescence. The HOMO and LUMO energy
levels of benzoic acid, propionic acid, acrylic acid, 1,3-dibenzoic
acid, and 2-aminobenzoic acid were estimated by computational
semiempirical (AM1) calculations.
Figure 10. Thermo gravimetric analysis overlay graph of dyes 1aꢀ1e.
Relative Fluorescence Quantum Yield. Quantum yields of
compounds 1aꢀ1e were determined by the comparative method
presented in Figures 3 and 4. The fluorescence intensity ratio
of the short emission band to the long emission band vs pH
curves for fluorescein derivatives 1a and 1d in aqueous solution
are given in Figure 5. It was observed that for compound 1a and
using known standards (fluorescein and anthracene) and formula
55,56
1
0
1
0
.
The quantum efficiency of compound 1a was found to be
.0879 at short wavelength and 0.624 at long wavelength. For dye
b and 1c, the quantum efficiency was found to be 0.224 and
.117, respectively. For compound 1d, the quantum efficiency
1
d, an exact opposite behavior of the ratiometric response was
located with respect to the change in pH value.
Fluorescence Properties of Fluorescein-Derivatives.
Fluorescent properties of fluorescein-derivatives are observed
to be truly controlled by the rate of photoinduced electron trans-
fer from the phthalein moiety (electron donor) to the singlet
excited state of the xanthene moiety (electron acceptor fluorophore)
in the excited state. The fluorescence properties of fluorescein are
highly dependable on oxidation potential and reduction potential
of the phthalein moiety. The electron density on the phthalein
ring influences the oxidation potential and reduction potential,
which finely controlled the fluorescence properties of substituted
was found to be 0.172 and 0.79, respectively, for short and long
wavelength emissions. For compound 1e, the quantum efficiency
was found to be 0.305. Relative fluorescence quantum yields are
summarized for compounds 1aꢀ1e in Table 2.
2
2
ϕ ¼ ϕ ðGrad =Grad Þðη =η
X
ST
Þ
ð1Þ
X
ST
X
ST
where ϕ = quantum yield of unknown sample; ϕ = quantum
X
ST
yield of standard used; Grad = gradient of unknown sample;
X
2
Grad = gradient of standard used; η
solvent for standard sample; and ηX = refractive index of solvent
for unknown sample.
= refractive index of
X
ST
5
0,51
52
2
fluorescein.
An example is well explained in the literature.
The presence of electron-withdrawing groups on the phthalein
moiety lowers the electron density controlling the fluorescence
Thermal Stability of Dyes. In order to give more insight into
the dyes 1aꢀ1e, the thermal studies of the compounds have been
carried out using thermal gravimetric techniques (TGA). The
thermogravimetric studies have been carried out in the tempera-
ture range 50ꢀ600 °C under nitrogen gas at a heating rate of
53,54
quantum efficiency of fluorescein derivatives.
It is known that dicarboxyfluorescein is highly fluorescent
ϕfl = 0.817), while disubstituted trimethyl ester is weakly fluo-
(
rescent (ϕ = 0.001). A similar phenomenon has also been
fl
53
ꢀ1
observed in the 6-carboxyfluorescein derivative (ϕ = 0.869).
10 °C min . The TGA results indicated that the synthesized
fl
Fluorescence quantum efficiency of fluorescent compounds is also
attributed to the energy values of HOMO and LUMO (Figure 6).
The dyes 1b, 1c, and 1e showed a single emission band, while
dyes are stable up to 300 °C. TGA revealed the onset decom-
position temperature (T ) of dyes 1aꢀ1e at 323, 300, 244, 335,
d
and 314 °C, respectively. Above 300 °C, the thermo gravimetric
1
a and 1d showed two emission bands. In the case of dual-
curve of the synthesized compounds show a major loss in weight.
emitting dyes, the relative fluorescence quantum efficiencies at
The comparisons of the T show that the thermal stability of
d
each emission wavelengths were calculated by using anthracene
1aꢀ1e decreases in the order 1d > 1a > 1e > 1b > 1c. The results
showed that synthesized dyes have good thermal stability, except
dye 1c, which showed a sharp decomposition point at 244 °C
and completely decomposed beyond 450 °C. However, dyes 1a,
1b, 1d, and 1e showed elongated decomposition behaviors and
55,56
and fluorescein as standards( Figure 7).
amino substituted derivative 1e, fluorescence quantum efficiency
for short wavelength emission was observed to be higher (ϕ1e436
.3057); the oxidation potential of the phthalein moiety was also
In the case of the
=
0
5
43
dx.doi.org/10.1021/jp2073123 |J. Phys. Chem. A 2012, 116, 536–545

The Journal of Physical Chemistry A
ARTICLE
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’
ASSOCIATED CONTENT
7
(
S
Supporting Information. Preparation, analysis, graph, and
b
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for HOMO and LUMO orbitals of each molecule. This material
is available free of charge via the Internet at http://pubs.acs.org.
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’
AUTHOR INFORMATION
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Corresponding Author
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E-mail: n.sekar@ictmumbai.edu.in or nethi.sekar@igmail.com.
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5
089–5093.
’
ACKNOWLEDGMENT
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