Novel coumarin-based pH sensitive fluorescent probes for the highly alkaline pH region

Article abstract of DOI:10.1016/j.dyepig.2020.108312

The design, synthesis and spectroscopic properties of novel polymerizable coumarin-based pH indicators are reported. They show a fluorescence response to pH in the highly alkaline pH region with calculated pK_a values in the range of 12.2–12.5. Covalent immobilization of the indicators to polymer supports provides novel pH-sensitive materials that possess excellent photostability. The materials are sensitive to pH in a similar range and can be applied in optical pH sensors for measuring pH in various alkaline media.

Full text of DOI:10.1016/j.dyepig.2020.108312

Journal Pre-proof
Novel coumarin-based pH sensitive fluorescent probes for the highly alkaline pH
region
T. Hien Nguyen, Tong Sun, Kenneth T.V. Grattan
PII:
S0143-7208(19)30581-9
DOI:
https://doi.org/10.1016/j.dyepig.2020.108312
DYPI 108312
Reference:
To appear in: Dyes and Pigments
Received Date: 15 March 2019
Revised Date: 24 February 2020
Accepted Date: 25 February 2020
Please cite this article as: Nguyen TH, Sun T, Grattan KTV, Novel coumarin-based pH sensitive
fluorescent probes for the highly alkaline pH region, Dyes and Pigments (2020), doi: https://
doi.org/10.1016/j.dyepig.2020.108312.
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Author statement
Thu Hien Nguyen: conceptualisation, planning of experiments, data analysis,
preparation of the manuscript.
Tong Sun: insightful comments and suggestions, preparation of the manuscript.
Kenneth T. V. Grattan: insightful comments and suggestions, preparation of the
manuscript.

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Novel coumarin-based pH sensitive fluorescent probes for the highly
alkaline pH region
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T. Hien Nguyen*, Tong Sun and Kenneth T.V. Grattan
Photonics and Instrumentation Research Centre, City University of London, London
EC1V 0HB, UK
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Email: hien.nguyen@city.ac.uk, Tel :+44(0)2070403257, Fax : +44(0)2070408568
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Abstract:
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The design, synthesis and spectroscopic properties of novel polymerizable coumarin-
based pH indicators are reported. They show a fluorescence response to pH in the
highly alkaline pH region with calculated pKa values in the range of 12.2 – 12.5.
Covalent immobilization of the indicators to polymer supports provides novel pH-
sensitive materials that possess excellent photostability. The materials are sensitive to
pH in a similar range and can be applied in optical pH sensors for measuring pH in
various alkaline media.
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Keywords: Fluorescent sensor, fluorescent polymer, high pH sensor, coumarin dye.
1. Introduction
The development of reliable techniques to monitor pH has received significant
attention because of the importance of pH measurement in various areas of scientific
research and other practical applications[1-8]. The determination of pH is routinely
performed using glass electrodes. However, glass electrodes have several
disadvantages and thus are not suitable for certain applications. They are bulky,
invasive and potentially create the risk of electric shock during in vivo measurements.
They show poor performance when measuring solutions of low ionic strength and
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suffer from surface potentials. In highly alkaline media, glass electrodes suffer from
chemical deterioration and are subject to large alkaline errors[9, 10].
pH sensing using optical methods provides an attractive alternative for measuring pH
due to the advantages offered in terms of immunity to electrical interference,
enhanced safety and the possibility of remote sensing. Most optical pH sensors
function through monitoring the changes in the absorbance or fluorescence properties
of certain pH sensitive indicators which are immobilized on/in proton-permeable solid
substrates/supports[11]. A number of pH indicators therefore have been designed or
investigated for this application. However, the majority of them were demonstrated to
work in the physiological/near neutral[12-16] or acidic pH region[5, 8, 11, 17, 18].
Materials that have been reported for use in the highly alkaline pH region are mainly
non-immobilized forms that are not ready for use in certain sensing applications[19,
20] or restricted to the absorbance/transmission based group[6, 21-23], which has
certain disadvantages compared to the fluorescence-based counterpart. In recent
years, a few fluorescent sensors for the alkaline pH region have been prepared[24-27].
However, the number of reports is still very limited. Considering the importance of
monitoring pH in highly alkaline media as occurring in numerous industrial
processes[9], scrubbers, waste water treatment plants and concrete structures[28, 29],
there is essentially a need for accurate, sensitive and reliable sensing materials
suitable for measurement of high basicity.
In this paper, the synthesis and characterization of novel polymerizable coumarins
bearing imidazolyl groups of which the deprotonation/protonation allows for the
determination of pH in the alkaline region of the pH scale are described and their
spectral and sensing properties are reported. Coumarins have been chosen because
they have many advantages including high fluorescence quantum yield, large Stokes
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shift and excellent photo-stability[30]. In addition to the development of appropriate
pH indicators, the choice of solid supports and the effective immobilization of the
indicators to the supports are also discussed in the paper as they are key factors in the
development of an optimum optical pH sensor since they govern the lifetime and
signal stability of the sensor. The fluorescence detection method was employed rather
than the simpler and more commonly-used method based on absorbance/transmission
measurements as fluorescent sensors are usually more precise, having less
interference from the light source, and offer higher sensitivity than their absorbance
counterparts[7, 31].
2. Experimental
2.1. General
All chemicals were of analytical grade, purchased from Sigma-Aldrich and were used
without further purification. All solvents used were of HPLC grade from Fisher
Scientific or Sigma-Aldrich. All aqueous solutions were prepared using distilled
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deionized water. H and 13C NMR spectra were recorded on a Bruker Avance 500
spectrometer. Mass spectra were run by electron ionisation (EI) mode on a Thermo
Finnigan MAT900xp mass spectrometer. IR spectra were recorded on a Bruker Alpha
Fourier Transform Infrared Spectrophotometer and were run neat. Melting points
were recorded using a Stuart SMP30 melting point apparatus and were uncorrected.
Elemental analyses were carried out at the Microanalytical Laboratory, Department of
Chemistry at University College London. Absorption and fluorescence measurements
of aqueous solutions containing fluorophores were carried out on a PerkinElmer
Lambda 35 spectrophotometer and
a
Horiba Jobin Yvon Fluoromax-4
spetrofluorometer system with FluorEssenceTM as driving software, respectively.
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Refractive indices were measured on an Abbe refractometer. Quantum yields of
fluorescence were determined using quinine sulfate as the standard (Φ = 0.55)[32,
33].
2.2. Synthesis of the fluorescent dyes
3-N-(Carbethoxy)aminophenol (1): 1 was prepared similarly to the method reported
in the literature[34]. To a two necked flask equipped with a condenser and a septum
were added 3- aminophenol (10.9 g, 100 mmol) and EtOAc (40 mL). The mixture
was heated to reflux for 30 min. Ethyl chloroformate (5.4 g, 4764 µL, 50 mmol) was
added via syringe over a 10 min period. The reaction mixture was left stirred for a
further 20 min then allowed to cool to room temperature to form a white precipitate.
The precipitate was removed by filtration and washed with EtOAc (50 mL) and
petroleum ether (50 mL). The combined filtrate was concentrated to give 1 as an off-
white solid which was further purified by recrystallization from toluene to afford 1
(8.4g, 93%) as white crystals, mp 92 oC [lit. mp 94-95 oC (benzene/cyclohexane)]; IR
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(neat) νmax (cm-1) 3294 (NH), 1684 (C=O), 1556, 1449, 1249, 1061; H-NMR (500
MHz, CDCl3) δ(ppm): 7.35 (s, 1H), 7.13 (t, 1H, J = 8.08), 6.69 (s, 1H), 6.64 (m, 1H),
6.57 (m, 1H), 6.33 (m, 1H), 4.23 (q, 2H, -CH2-, J=7.12 Hz), 1.30 (t, 3H, -CH3, J =
7.12); MS (EI): Calcd. m/z = 181.07334 (C9H11O3N). Found m/z = 181.07296 (M+).
4-Chloromethyl-7-N-(Carbethoxy)aminocoumarin (2): H2SO4 (80%, 40 mL) was
pre-cooled in an ice bath. 1 (1.8 g, 10 mmol) was added, followed by ethyl 4-
chloroacetoacetate (2.3 g, 1906 µL, 14 mmol) in portions. The mixture was stirred at
room temperature under Ar for 19 h, after which it was poured into ice-water (50 mL)
and left stirred for a further 30 min. The white precipitate formed was filtered, washed
with H2O and recrystallized from EtOH to afford 2 (2.0 g, 71%) as fine needles, mp
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242 C (decomp.) [lit.[35] mp 247 C (MeOH)]; IR (neat) νmax (cm-1) 3280 (NH),
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3084, 2992, 2929, 1704 (C=O), 1619, 1588, 1520, 1404, 1332, 1204, 1075; H-NMR
(500 MHz, CDCl3) δ(ppm): 7.59 (d, 1H, H5, J5,6=8.7 Hz), 7.50 (d, 1H, H8, J8,6=2.14
Hz), 7.36 (dd, 1H, H6, J6,5=8.7 Hz, J6,8=2.14), 6.82 (s, 1H, NH), 6.45 (s, 1H, H3),
4.65 (s, 2H, -CH2Cl), 4.27 (q, 2H, OCH2-, J = 7.13), 1.34 (t, 3H, -CH3, J = 7.13); MS
(EI): Calcd. m/z = 281.04494 (C13H12O4NCl). Found m/z = 281.04550 (M+).
4-Chloromethyl-7-aminocoumarin (3): 2 (563.4 mg, 2 mmol) was suspended in a
mixture of concentrated H2SO4 (1.7 mL) and glacial acetic acid (1.7 mL). The mixture
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was heated to 125 C for 2h. After cooling to room temperature, the yellow solution
was poured into H2O and a voluminous precipitate was formed. 4 M NaOH aqueous
solution was added to the mixture to bring pH to ~ 9. The yellow precipitate was
filtered, washed with H2O, dried in vacuo to afford 3 (366 mg, 87%) as a pale yellow
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solid which was used for synthesis without further purification, mp 182 C [lit.[35]
mp 187 oC]; IR (neat) νmax (cm-1) 3445 (NH2), 3352 (NH2), 1686 (C=O), 1601 (NH2),
1548, 1404, 1263, 1210, 1145; 1H-NMR (500 MHz, DMSO) δ(ppm): 7.47 (d, 1H, H5,
J5,6=8.7 Hz), 6.58 (dd, 1H, H6, J6,5=8.7 Hz, J6,8=2.00), 6.43 (d, 1H, H8, J8,6=2.00 Hz)
6.19 (s+s, nonres., 3H, -NH2 + H3), 4.86 (s, 2H, -CH2Cl); MS (EI): Calcd. m/z =
209.02381 (C10H8O2NCl). Found m/z = 209.02417 (M+).
7-amino-4-((2-methyl-4-nitro-1H-imidazol-1-yl)methyl)-coumarin (4): NaH in
mineral oil (60%, 60 mg) was added to a solution of 2-methyl-4-nitroimidazole (203
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mg, 1.6 mmol) in DMF (6 mL). The mixture was heated to 100 C for 10 min and
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then cooled down to 60 C, after which 3 (314 mg, 1.5 mmol) dissolved in DMF (6
mL) was added. The reaction mixture was left stirring at the same temperature for 18
h then poured into ice. The yellow precipitate formed was collected by filtration,
washed with water and recrystallized from EtOH to afford 4 (390 mg, 87%) as
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yellow fine crystals, mp 298-300 C (decomp.); IR (neat) νmax (cm-1) 3430 (NH2),
3324 (NH2), 3221, 3144, 1695 (C=O), 1595 (NH2), 1542, 1498, 1402, 1328, 1290,
1132; 1H-NMR (500 MHz, DMSO) δ(ppm): 8.37 (s, 1H, 5-H-imidazole), 7.44 (d, 1H,
H5, J5,6=8.69 Hz), 6.61 (dd, 1H, H6, J6,5=8.69 Hz, J6,8=2.14), 6.45 (d, 1H, H8,
J8,6=2.14 Hz) 6.27 (s, 2H, -NH2), 5.51 (s, 2H, -CH2-), 5.05 (s, 1H, H3), 2.31 (s, 3H, -
CH3); 13C-NMR (DMSO) δ(ppm): 160.7 (C2), 155.9 (C9), 154.0 (C7), 151.1 (4-C-
imidazole), 146.1 (C4), 125.7 (C5), 123.1 (5-C-imidazole), 111.7 (C6), 106.2 (C10),
104.5 (C3), 99.0 (C8), 46.6 (-CH2- ), 12.8 (-CH3); MS (EI): Calcd. m/z = 300.08531
(C14H12O4N4). Found m/z = 300.08584 (M+); Elem. Anal. Calcd. for C14H12O4N4
(300.27): C 56.00 , H 4.03, N 18.66. Found: C 56.72 , H 4.50, N 18.19.
7-(4-vinylbenzylamino)-4-((2-methyl-4-nitro-1H-imidazol-1-yl)methyl)-coumarin
(5): A mixture of 4 (300 mg, 1 mmol), vinylbenzylchloride (155 µL, 168 mg, 1.1
mmol, 1 mmol), potassium carbonate (401 mg, 2.9 mmol), posstasium iodide (49 mg,
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0.3 mmol) in dry MeCN (20 mL) was heated under argon at 80 C for 2 days. After
cooling to room temperature, H2O was added to dissolve the inorganic salts. The
mixture was filtered to remove insoluble materials. EtOAc was then added. The
organic phase was washed with H2O (2 x 50 mL) and saturated aqueous NaCl (50
mL), dried over MgSO4, filtered, and concentrated in vacuo. The resulting orange-red
residue was chromatographed on silica gel using CH2Cl2-EtOAc (4:6, v/v) as eluent to
give an orange solid which was further purified by recrystallization from ethanol to
afford 5 (40 mg, 10 %) as a yellow-orange solid, mp 194 C; IR (neat) νmax (cm-1)
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3335 (NH), 3144, 1694 (C=O), 1601, 1540, 1500, 1404, 1321, 1291, 1148; H-NMR
(500 MHz, CDCl3) δ(ppm): 7.69 (s, 1H, 5-H-imidazole), 7.41 (d, 2H, aromaticH.
J=8.13 Hz), 7.30 (d, 2H, aromaticH J=8.15 Hz), 7.20 (d, 1H, H5, J5,6=8.72 Hz), 6.71
(dd, 1H, CH=CH2, J=10.91 Hz, J=17.60 Hz), 6.57 (dd, 1H, H6, J6,5=8.72 Hz,
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J6,8=2.36 Hz), 6.53 (d, 1H, H8, J8,6=2.32 Hz), 5.75 (d, 1H, CH=CHaHb J=17.61 Hz),
5.43 (s, 1H, H3), 5.26 (d, 1H, CH=CHaHb ,J=10.90 Hz), 5.17 (s, 2H, C4CH2-), 4.85
(t, 1H, -NH-, JNH-CH2 = 5.52 Hz), 4.41 (d, 2H, CH2NH-, JCH2-NH = 5.55 Hz), 2.44 (s,
3H, -CH3); 13C-NMR (CDCl3) δ(ppm): 156.3 (C2), 155.0 (2-C- imidazole), 154.0
(C9), 147.7 (C7), 141.1 (C4), 140.0 (4-C-imidazole), 137.3 (CCH2NH-), 136.2
(CH=CH2), 128.0 ((CCH=CH2), 127.6 (aromaticC), 126.8 (aromaticC), 123.7 (C5),
119.9 (5-C-imidazole), 114.8 (CH=CH2), 107.6 (C3), 107.1 (C10), 99.1 (C8), 47.5 (-
CH2NH-), 46.9 (C4CH2-), 13.1 (-CH3); MS (EI): Calcd. m/z = 416.14791
(C23H20O4N4). Found m/z = 416.14827 (M+); Elem. Anal. Calcd. for C23H20O4N4
(416.43): C 66.34 , H 4.89, N 13.45. Found: C 65.76 , H 4.66, N 12.89.
7-acrylamido-4-((2-methyl-4-nitro-1H-imidazol-1-yl)methyl)-coumarin (6):
Acryloyl chloride (76 µL, 75 mg, 0.93 mmol) in dry THF (4 mL) was added slowly to
a suspension of 4 (112 mg, 0.373 mmol) in dry THF (10 mL) under argon. The
reaction mixture was left stirred at room temperature overnight. The resulting
precipitate was collected by filtration, washed with THF, H2O and ethyl acetate then
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dried in vacuo to afford 6 (124 mg, 94%) as a pale yellow solid, mp 291 C
(decomp.); IR (neat) νmax (cm-1) 3301, 1695 (C=O), 1608, 1501, 1414, 1332, 1285,
1211, 1188, 1141; 1H-NMR (500 MHz, DMSO) δ(ppm): 10.65 (s, 1H, -CONH-), 8.38
(s, 1H, 5-H-imidazole), 7.90 (d, 1H, H8, J8,6=1.68 Hz), 7.77 (d, 1H, H5, J5,6=8.70 Hz),
7.59 (dd, 1H, H6, J6,5=8.72 Hz, J6,8=1.71), 6.46 (dd, 1H, CH=CH2, J=10.08 Hz,
J=16.91 Hz), 6.33 (dd, 1H, CH=CHaHb, J=16.95 Hz, J=1.54 Hz), 5.85 (dd, 1H,
CH=CHaHb, J=10.19 Hz, J=1.43 Hz), 5.61 (s, 2H, -CH2-), 5.48 (s, 1H, H3), 2.35 (s,
3H, -CH3); 13C-NMR (DMSO) δ(ppm): 163.8 (C=ONH-), 160.7 (C2), 153.8 (C9),
149.9 (C7), 145.8 (4-C-imidazole), 142.8 (C4), 131.3 (CH=CH2), 129.0 (CH=CH2),
125.2 (C5), 122.7 (5-C-imidazole), 115.5 (C6), 112.0 (C10), 110.1 (C3), 106.1 (C8),
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46.3 (-CH2- ), 12.5 (-CH3); MS (EI): Calcd. m/z = 354.09587 (C17H14O5N4). Found
m/z = 354.09630 (M+); Elem. Anal. Calcd. for C17H14O5N4 (354.32): C 57.63 , H
3.98, N 15.81. Found: C 56.14 , H 3.61, N 14.05.
4-Chloromethyl-7-bromocoumarin (7): H2SO4 (80%, 40 mL) was pre-cooled in an
ice bath. 3-bromophenol (1.73 g, 10 mmol) was added, followed by ethyl 4-
chloroacetoacetate (2.3 g, 1906 µL, 14 mmol) in portions. The mixture was stirred at
r.t under Ar for 22 h, after which it was poured into ice-water (50 mL) and left stirred
for a further 30 min. The white precipitate formed was filtered, washed with H2O,
dried over phosphorus pentoxide and recrystallized from EtOH to afford 7 (1.87 g,
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68%) as a white solid, mp 211 C; IR (neat) νmax (cm-1) 3065, 1736 (C=O), 1598,
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1395, 1271, 1243, 1173, 1151, 1081; H-NMR (500 MHz, CDCl3) δ(ppm): 7.56 (d,
1H, H8, J8,6=1.84 Hz), 7.54 (d, 1H, H5, J5,6=8.51 Hz), 7.47 (dd, 1H, H6, J6,5=8.37 Hz,
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J6,8=1.68), 6.58 (s, 1H, H3), 4.64 (s, 2H, -CH2Cl); C-NMR (DMSO) δ(ppm): 159.1
(C2), 153.8 (C4), 150.2 (C9), 127.5 (C6), 126.9 (C8), 125.1 (C7), 119.8 (C5), 116.4
(C10), 115.8 (C3), 41.1 (-CH2-); MS (EI): Calcd. m/z = 271.89501 (C10H6O2BrCl).
Found m/z = 271.89465 (M+). Elem. Anal. Calcd. for C10H6O2BrCl (273.51): C 43.91,
H 2.21. Found: C 43.35 , H 2.19.
7-bromo-4-((2-methyl-4-nitro-1H-imidazol-1-yl)methyl)-coumarin (8): NaH in
mineral oil (60%, 80 mg) was added to a solution of 2-methyl-4-nitroimidazole (254.2
mg, 2 mmol) in DMF (8 mL). The mixture was heated to 110 oC for 10 min and then
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cooled down to 60 C, after which 7 (547.0 mg, 2 mmol) suspended in DMF (8 mL)
was added. The reaction mixture was left stirring at the same temperature for 20 h
then poured into ice. The precipitate formed was collected by filtration, washed with
water, dried and recrystallized from EtOH to afford 8 (538 mg, 74%) as an off-white
solid, mp 289-291 oC (decomp.); IR (neat) νmax (cm-1) 3135, 1738 (C=O), 1597, 1541,
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1498, 1398, 1342, 1299, 1244, 1171, 1147; H-NMR (500 MHz, DMSO) δ(ppm):
8.36 (s, 1H, 5-H-imidazole), 7.81 (d, 1H, H8, J8,6=1.90 Hz), 7.73 (d, 1H, H5, J5,6=8.53
Hz), 7.66 (dd, 1H, H6, J6,5=8.52 Hz, J6,8=1.92 Hz), 5.68 (s, 1H, H3), 5.63 (s, 2H, -
CH2-), 2.35 (s, 3H, -CH3); 13C-NMR (DMSO) δ(ppm): 158.8 (C2), 153.4 (C9), 149.5
(4-C-imidazole), 145.9 (C4), 145.8 (2-C-imidazole), 127.6 (C6), 126.2 (C5), 125.4
(C7), 122.6 (5-C-imidazole), 119.8 (C8), 116.4 (C10), 112.8 (C3), 46.3 (-CH2-), 12.5
(-CH3); MS (EI): Calcd. m/z = 362.98492 (C14H10O4N3Br). Found m/z = 362.98521
(M+); Elem. Anal. Calcd. for C14H10O4N3Br (364.15): C 46.18 , H 2.77, N 11.54.
Found: C 45.19 , H 2.36, N 10.88.
7-vinylphenyl-4-((2-methyl-4-nitro-1H-imidazol-1-yl)methyl)-coumarin (9):
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was prepared from 8 via a Suzuki coupling reaction[36-38] with vinylphenylboronic
acid. A mixture of 8 (400.6 mg, 1.1 mmol), 4-vinylphenylboronic acid (244.2 mg,
1.65 mmol, 1.5 mol equiv), potassium carbonate (570 mg, 4.125 mmol, 3.75 mol
equiv) and dioxane (11 mL) was stirred in a two necked flask at room temperature
under argon for 0.5 h. Tetrakis(triphenylphosphine)palladium(0) (63.6 mg, 0.055
mmol, 5 mol %) was added. A condenser was fitted and the flask was evacuated and
filled with argon three times before being heated to 80 oC. The reaction was left at 80
oC in the dark for 41 h. After cooling to room temperature, EtOAc was added. The
organic phase was washed with water (3 × 50 mL), saturated aqueous NaCl (50 mL),
dried over MgSO4, filtered, concentrated in vacuo to give the crude product which
was purified by flash chromatography on silica gel with EtOAc-petroleum ether (95:5,
v/v) as eluent to give 9 as an orange solid, which was further purified by
recrystallization from EtOH-EtOAc (5:1, v/v) to give orange crystals (176 mg, 41%),
mp 215 C; IR (neat) νmax (cm-1) 3147, 1716 (C=O), 1612, 1538, 1496, 1395, 1322,
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1290, 1160; 1H-NMR (500 MHz, DMSO) δ(ppm): 8.40 (s, 1H, 5-H-imidazole), 7.89-
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7.78 (m, 5H, H5, H6, H8, aromaticH), 7.62 (d, 2H, aromaticH J=8.34 Hz), 6.80 (dd,
1H, CH=CH2, J=10.98 Hz, J=17.67 Hz), 5.94 (d, 1H, CH=CHaHb, J=17.70 Hz), 5.68
(s, 2H, -CH2-), 5.63 (s, 1H, H3), 5.34 (d, 1H, CH=CHaHb ,J=11.00 Hz), 2.37 (s, 3H, -
CH3); 13C-NMR (DMSO) δ(ppm): 159.5 (C2), 153.6 (C9), 149.8 (4-C-imidazole),
145.9 (C4), 145.8 (C7), 143.6 (2-C-imidazole), 137.6 (CC7), 137.2 (CCH=CH2),
136.0 (CH=CH2), 127.3 (aromaticC.), 126.9 (aromaticC.), 125.1 (C5), 122.7 (C6),
122.6 (5-C-imidazole), 116.1 (C10), 115.3 (CH=CH2), 114.1 (C8), 112.0 (C3), 46.4 (-
CH2-), 12.5 (-CH3); MS (EI): Calcd. m/z = 387.12136 (C22H17O4N3). Found m/z =
387.12164 (M+); Elem. Anal. Calcd. for C22H17O4N3 (387.39): C 68.21 , H 4.42, N
10.85. Found: C 66.92 , H 4.14, N 10.15.
2.3. Preparation of polymers
The compositions of the different polymers are given in Table 1.
Polymer
Fluorescent dye
Type /mmol
MAA
/mmol
0.1
BAP
/mmol
0.5
P84
/mmol
0.02
DMF
/µL
PVIC
PAIC
PSIC
VIC (5)
AIC (6)
SIC (9)
0.05
0.05
0.05
500
500
500
0.1
0.5
0.02
0.1
0.5
0.02
239
240
241
242
243
244
245
Table 1. Composition of polymers prepared using different coumarin-based pH
indicators. MAA: methylmethacrylate, BAP: 1,4-bis(acryloyl)piperrazine, P84:
Pluronic P84.
Coumarin, methacrylic acid, 1,4-bis(acryloyl)piperrazine and pluronic P84 were
weighed into a borosilicate glass vial and dissolved in dimethylformamide.
Azobis(isobutyronitrile) (AIBN) (5 mg) was then added. The vial was placed in a
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sonicating water bath until AIBN was fully dissolved, then purged thoroughly with
argon for about 2 min before being tightly capped and sealed. Polymerization was
o
carried out at 80 C in the dark for approximately 20 h. The resulting hard bulk
polymer was then hand ground with a mortar and pestle until fine particles were
obtained. The polymer particles were washed to remove unreacted materials by
repeated incubation in MeOH (50 mL solvent each), centrifugation and re-suspension
(4×0.5h incubations), followed by the same procedure with H2O (2×0.5 h incubations)
and finally on a sintered filter with MeOH (50 mL). After washing, polymer particles
were dried in vacuum and stored in the dark until use.
2.4. Fluorescence measurements
Emission spectra of fluorescent indicators and polymers were recorded using a 4 mL
(1×1×4 cm) quartz cuvette. Slit widths were adjusted to give emission below the
saturating limit (1000000 cps) of the detector, and then the same settings were used
for a complete set of experiments.
0.8 mM indicator stock solutions were prepared by dissolving 2x10-3 mmol of each
indicator in 2.5 mL EtOH. PVIC (6.3 mg/mL, containing 2.5 µmol coumarin/mL),
PAIC (5.7 mg/mL) and PSIC (5.8 mg/mL) stock solutions were prepared in deionized
distilled water.
50 mM phosphate at various pH values ranging from 10 to 13.6 was prepared by
dissolving Na2HPO4 (1.77 g, 12.5 mmol) in distilled deionized water (240 mL) in a
conical flask. The buffer was adjusted to the desired pH with aqueous NaOH. The
solution was then transferred to a 250 mL volumetric flask and made up to the volume
with distilled water.
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20 µL of each coumarin stock solution or 50 µL of each polymer stock solution was
added to 3 mL of buffer in a cuvette, followed by measurement of emission spectra.
The cuvette was shaken vigorously before each measurement. All samples were
prepared in duplicate or triplicate and the data presented were average values.
It should be noted that phosphate does not act as a good buffer at pH values higher
than 12 -12.5. However, phosphate was used for all pH to avoid any differences in
fluorescence caused by the difference in buffer composition. It has also been
confirmed by measuring pH of the solutions before and after adding the
dyes/polymers that a small amount of either the free dyes or the polymers does not
cause any change in the pH values of the solutions.
3. Results and discussion
3.1. Design and synthesis of fluorescent pH indicators
Although there has been a variety of pH indicators, both commercially available
including azo dyes[6, 21, 23, 39, 40], trinitrobenzensulfonic acid[28], thymol blue[29,
41-43], cresol red[41], phenolphthalein[44], aniline blue[45], rhodamine 19[46],
Victoria blue[47] etc. as well as reported in the literature such as fluorescent Schiff
bases[48], porpholactones[49] or a benzo[de]anthracen-7-one derivative[31] , that are
known to be useful for the spectroscopic determination of high pHs, the majority of
them are absorptive dyes rather than fluorescent dyes and only a few meet the
requirements of being stable for a long period of time in highly alkaline media,
possessing a pKa in the range between 11-12.5, having reasonable fluorescence
quantum yield and good photostability and bearing one or more functional chemical
groups suitable for covalent immobilization of the indicator. Coumarins have been
employed and investigated in this work as they are widely used as laser dyes for
12

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single-molecule fluorescence and so they are ‘tried and tested’ in terms of the key
property of being photo-stable[50, 51]. It has also been reported that a coumarin with
an imidazole substituent shows response to pH in the alkaline region [19]. The design
of the indicator dyes was tailored based on this core structure and suitable functional
group(s) for immobilization of the indicator to a substrate.
Having created appropriate indicator dyes, it is equally important to perform an
effective immobilization of the dyes to a suitable solid support/substrate. Prior work
has shown that poor immobilization results in dye leaching and consequently a
drifting of the calibration of the probe, which leads to the gradual breakdown of its
useful sensing ability[11]. Among several widely used immobilization methods such
as absorption or entrapment[52-54], layer-by-layer (LbL) electrostatic self-assembly
[55, 56] and covalent binding[21, 57-60], the covalent binding method is believed to
produce more reliable and durable sensors, as the indicators are virtually bonded to
the substrate therefore they are unlikely to leach out under normal conditions,
although the fabrication process is relatively complicated and time-consuming.
Regarding solid supports, various materials have been employed as the supports for
dye immobilization including cellulose membrane[21, 23, 57, 61], silica sol-gels[15,
62] and synthetic organic polymers[39, 60] with cellulose and sol-gels being more
commonly used than the other due to the optical transparency of their matrices and in
many cases, the convenience of attachment process. However, cellulose degrades
easily in highly alkaline media[22, 39] and sol-gels are completely deteriorated at pH
higher than 10 within relatively short time. Therefore, they are not suitable for this
study, leaving synthetic polymers the favourable option. The indicator dye can be
connected into polymer matrix by either co-polymerisation of the dye monomer and
13

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344
appropriate co-monomer(s) or by attaching the reactive dye to a readily prepared
polymer. In this work, the former method was used because it is easier to design
different polymer matrices and control their properties to meet the requirements of the
sensor in terms of polymer rigidity, hydrophilicity and optical density by using
different mixtures of co-monomers.
To meet the desired requirements discussed above, the fluorescent dyes used in this
work were designed to include a polymerizable group and the coumarin core structure
with an imidazole substituent. The linkage between the polymerizable group and the
coumarin should be stable enough to sustain strongly alkaline media. Three novel
polymerisable coumarins: vinylbenzylamino imidazolylmethyl coumarin (VIC, 5),
acrylamido imidazolylmethyl coumarin (AIC, 6) and styryl imidazolylmethyl
coumarin (SIC, 9) were prepared in multiple steps starting from commercially
available phenolic compounds as outlined in Scheme 1. The Pechmann reaction of
phenols with a β-carbonyl ester is a versatile approach for the synthesis of 4-
substituted coumarins. 2 and 7 were prepared similarly to the method reported in the
literature[35]. The substitution of chlorine for an imidazolyl group was achieved by
performing a reaction with 2-methyl-4-nitroimidazole, following the method
described by Al-Soud et al. [63]. Polymerizable groups were introduced into the
coumarin structures either via a Suzuki coupling[36-38] of the Br-substituted
coumarin 8 with vinylphenylboronic acid using K2CO3 in dioxane as a base/solvent
mixture or by reacting the amine substituted coumarin 4 with acryloyl chloride to give
the amide 6 or with 4-vinylbenzylchloride to give the secondary amine 5.
14

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369
Scheme 1. Preparation of fluorescent monomers. (a) ClCOOEt, EtOAc, 100 oC, 93%;
(b) ethyl 4-chloroacetoacetate, H2SO4, H2O, r.t, 19 h, 71%: (c) conc.H2SO4, glacial
AcOH, 125 oC, 2 h, 87%; (d) NaH (60%), 2-methyl-4-nitroimidazole, DMF, 100 oC to
60 oC, 18 h, 87%; (e) vinylbenzylchloride, K2CO3, KI, MeCN, 80 oC, 2 days, 10%; (f)
CH2=CHCOCl, dry THF, r.t., 12 h, 94%; (g) ethyl 4-chloroacetoacetate, H2SO4, H2O,
r.t, 22 h, 68%; (h) NaH (60%), 2-methyl-4-nitroimidazole, DMF, 110 oC to 60 oC, 20
h, 74%; (i) CH2=CHC6H4B(OH)2, Pd(PPh3)4, K2CO3, dioxane, 80 oC, 41 h, 41%.
3.2. Spectroscopic properties and pKa values of the free indicators
In neutral aqueous solution (with the addition of 1.5% EtOH to solubilize the
compounds), the absorption spectra of VIC (5) and AIC (6) show only one main
absorption band of each dye in the UV region, at 370 nm and 330 nm, respectively,
15

370
371
(Fig. 1) whereas that of SIC (9) shows two main bands, at 266 nm with weaker
absorbance and 334 nm with higher absorbance.
0.014
0.214
0.194
0.174
0.154
0.134
0.114
0.094
0.074
0.054
0.034
0.012
0.01
0.008
0.006
0.004
0.002
0
240
290
340
390
440
490
540
590
Wavelength (nm)
372
373
374
375
Fig.1. Absorption (left) and emission (right) spectra of VIC (5) (solid lines), SIC (9)
(broken lines) and AIC (6) (dotted lines) (5 µM) in H2O. Emission spectra recorded
with λex = 330 nm for SIC and AIC and 370 nm for VIC.
16

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380
381
382
383
384
385
386
387
388
Table 2 Spectral data and pKa values of the fluorescent dyes.
Emission spectra for each compound recorded in the same solvent using excitation at
the absorbance maxima include only one band in the 415-470 nm region. VIC absorbs
and emits at longer wavelengths compared to AIC and SIC but its fluorescence
quantum yield is fairly similar to that of AIC and much lower than that of SIC (Table
2). This difference in fluorescence can be explained by the fact that coumarin
molecules have close lying π,π* and n,π* states which are easily perturbed by changes
in solvents, substituents and other factors[64-66], and hence the excited state S1
probably has both π,π* and n,π* character. It is proposed that in VIC, a n,π* state may
lie below the π,π* state and at a low energy level, due to the great conjugation of the
lone-pair electrons of the amine nitrogen at the 7 position with the π system. This
degree of conjugation is decreased in AIC where the amine nitrogen is converted to an
amide counterpart, which may result in the low lying excited state shifting to higher
Dye
Solvent
UV max
(nm)
Emission
max (nm)
pKa
Φ (%)
VIC (5)
VIC (5)
AIC (6)
AIC (6)
SIC (9)
SIC (9)
EtOH
H2O
371
370
334
330
335
334
456
470
407
416
448
458
-
2.6
0.8
1.7
0.6
12.48 ± 0.06
EtOH
H2O
-
12.22 ± 0.07
-
EtOH
H2O
25.0
2.0
12.40 ± 0.24
389
390
energy. In SIC where the π-electron system of coumarin is enhanced by the
substituted styryl group, the π,π* state may lie below the n,π* state, as shown in Fig.
17

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400
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402
403
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405
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409
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415
2. This could account for the lower quantum yields of VIC and AIC compared to SIC,
n←π* transitions being generally less intense than π←π* transitions because they
characteristically have longer lifetimes which enhance intersystem crossing[67, 68].
The red-shifted fluorescence of VIC with respect to AIC and SIC may be due to the
smaller HOMO-LUMO gap.
It is also noted that all dyes exhibit very large Stokes shifts (the difference in
wavelength between the absorption and the fluorescence spectral peaks) of 100 nm for
VIC, 86 nm for AIC and 124 nm for SIC, which is very important for sensing
applications since this minimizes the interference of the excitation light with the
fluorescence emission.
NO2
NO2
N
NO2
N
N
N
N
N
O
N
O
O
N
O
O
O
O
H
H
AIC
VIC
SIC
E
S*(π,π*)
S*(n,π*)
S*(n,π*)
S*(π,π*)
S*(π,π*)
S*(n,π*)
F
F
F
So
So
So
Fig. 2 Possible energy diagrams for VIC, AIC and SIC. So: ground state, S*: excited
single state, F: fluorescence.
In aqueous solution, all the dyes show no change in the fluorescence properties in the
neutral and near neutral pH region (data not shown) and a decrease in fluorescence
18

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440
intensity with increasing pH in the alkaline region (Fig.3). This can be attributed to
the equilibrium between the protonated and deprotonated forms of the imidazolyl
group as shown in Scheme 2. The protonated form is fluorescent and the deprotonated
form is much less so. Therefore, the fluorescence intensities of the dyes are lower at
higher pH values.
The deprotonation of the imidazolyl group at high pH values was further confirmed
by 13C NMR titrations of VIC, AIC and SIC with sodium methoxide (MeONa), a very
strong base, in DMSO-d6. Initially, the 13C spectrum of each fluorophore in DMSO-d6
(0.1 M concentration) was recorded. One mole equivalent of sodium methoxide in
DMSO-d6 was then added to each solution and 13C NMR spectra were recorded again.
The addition of MeONa resulted in chemical shifts of all carbon nuclei. However, the
largest changes in shift were for the carbon nuclei attached to the only proton of the
imidazolyl group (C5-imidazole) with changes of up to 1.71 ppm for the titration of
VIC and 1.63 ppm and 1.47 ppm respectively for the titrations of AIC and SIC. These
results confirm that the carbon nuclei C5 of the imidazolyl group is involved directly
in the interactions with the base through deprotonation. It has been reported that
imidazole is a very weak acid with pKa = 14.5[69]. However, in the presence of a
strong electron acceptor – NO2 group, its acidity increases and pKa can be shifted to a
lower value.
Scheme 2. Protonation-deprotonation equilibria of the fluorescent indicators in
aqueous solution.
19

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465
It has been proposed that the difference in fluorescence intensity between two forms
is due to photoinduced electron transfer (PET) between the imidazole anion ( proton
receptor) and coumarin (fluorophore)[19, 70]. At high pH, the electron-enriched
imidazole anion quenches the fluorescence of the coumarin through PET. At lower
pH, the presence of low concentration of hydrogen ions causes protonation of C5-
imidazole, resulting in PET from the imidazole to coumarin being hindered and hence
the fluorescence intensity increases.
The equilibrium between the protonated (HA) and deprotonated (A-) forms of the
indicators can be written in short as:
HA ↔ H + + A−
The relationship between the protonation state of the indicator and the pH is governed
by the Henderson-Hasselbalch equation:
−
[
]
A
pH = pKa + log
(1)
[
HA
]
where [A¯ ] and [HA] are the concentrations of the deprotonated and protonated forms
of the indicator and pKa is the acid-base constant. [A¯ ] and [HA] are related to
fluorescence intensities by [A¯ ] = F – Fmax and [HA] = Fmin – F where F is a
measured fluorescence intensity of the system, Fmax is the fluorescence intensity of
the fully protonated system and Fmin is the fluorescence intensity of the deprotonated
system. The expressions are then substituted into Equation 1 to provide Equation 2.
F − Fmax
pH = pKa + log
(2)
Fmin − F
Equation 2 can be re-written in terms of F to give
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484
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487
488
489
490
)
Fmax + Fmin ×10( pH − pK
a
F =
(3)
10( pH − pK ) +1
a
This results in an ‘S-shaped’ relation of the fluorescence intensity versus pH graph,
centered on the pKa value, which is the pH where 50% of the dye population in
solution is protonated.
The calculation of the pKa values for the indicators was performed based on the
fluorescence intensities at a fixed maximum emission wavelength at different pH
values using the model for nonlinear fitting method according to Equation 3. The data
obtained are summarized in Table 2. The pKa values for the three dyes are very
similar, suggesting that the substitution at the 7 position of the coumarin has no
2.E+06
1.8E+06
1.E+06
significant effect on the protonation-deprotonation of the imidazolyl group.
a)
1.E+06
1.6E+06
1.E+06
pKa = 12.48 ± 0.06
8.E+05
Increasing pH
6.E+05
1.4E+06
4.E+05
2.E+05
1.2E+06
0.E+00
10 10.5 11 11.5 12 12.5 13 13.5 14
1.0E+06
pH
8.0E+05
6.0E+05
4.0E+05
2.0E+05
0.0E+00
400 420 440 460 480 500 520 540 560 580 600
Wavelength (nm)
1.E+06
1.4E+06
b)
1.E+06
1.2E+06
1.0E+06
8.0E+05
6.0E+05
4.0E+05
2.0E+05
0.0E+00
8.E+05
6.E+05
4.E+05
2.E+05
pKa = 12.22 ± 0.07
Increasing pH
10 10.5 11 11.5 12 12.5 13 13.5 14
pH
350 370 390 410 430 450 470 490 510 530 550
Wavelength (nm)
21
1.E+06
1.4E+06
1.2E+06
1.E+06
c)
8.E+05
6.E+05
4.E+05
pKa = 12.40 ± 0.24
Increasing pH

491
492
493
494
495
496
497
498
499
500
501
502
Fig 3. Emission spectra of VIC (a), AIC (b) and SIC (c) at pH from 10.0 to 13.6. The
insets show the titration plots at emission maxima .
c).
λex = 370 nm (a), 330 nm (b and
503
504
505
506
507
508
509
510
511
512
513
514
515
3.3. Polymer preparation
Polymers were prepared in dimethylformamide (DMF) by free-radical polymerization
using azobis(isobutyronitrile) as initiator (refer to Table 1 for the compositions of the
different polymers). These were prepared as monolithic polymers and ground into
small particles, since these are easier to produce than spherical beads which can be
made by precipitation polymerisation and since a minimal amount of solvent is
required. There is no problem with scattering from the irregular-shaped particles
because with the coumarins used here the Stokes shift is sufficient that emission can
be measured well above the scattered wavelengths. Since the materials were designed
to work in aqueous environments, 1,4-bis(acryloyl)piperrazine was used as the cross-
linker as it is water soluble, hence producing polymers with a more hydrophilic
backbone. Methacrylic acid was included as a co-monomer so that the polymers
should not be too rigid and it can also increase the hydrophilicity of the polymers.
22

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522
523
524
525
526
527
528
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530
531
532
533
534
535
536
537
538
539
540
Pluronic P84 was added to increase the surface area and porosity. It is important that
the polymer used has the right molar ratio of cross-linker to monomers since lower
cross-linking gives insufficiently robust polymer and higher cross-linking gives too
rigid polymer, hence preventing the diffusion of hydrogen ions from surrounding
environments to the fluorescent receptor. After trying and testing different ratios in
terms, it was found that a molar ratio of cross-linker to monomers of about 10:3-4
produced good polymers for this application. The molar amount of fluorescent
monomer used was fixed at 1:10 of the cross-linker. More fluorescent monomer
would be expected to give a stronger fluorescent signal but too high a concentration of
fluorophore could also result in fluorescence quenching by the inner filter effect.
3.4. Photostability
Photostability is one of the critical properties of fluorescent materials used for sensor
applications. It is proposed that the substitution at the 7 position of the coumarin has
no significant effect on the photostability of the coumarin core structure.
Photostability was tested with one of the polymers PVIC by holding a layer consisting
of 4 mg of the polymer particles in between a quartz disc (6 mm dia.) and a nylon
membrane (20 µm pores, GE), which was kept in place by an O-ring and coupled into
the fluorimeter through a dichroic mirror using a fiber bundle. The excitation light (at
a wavelength of 375 nm) was launched to the distal end of the probe illuminating the
polymer with light from the intense, high power Xe lamp of the fluorimeter
continuously for 1 h. The fluorescence intensity data from the polymer were collected
over that period and displayed. Fig.4 shows the fluorescence intensities of the
polymer in dry state and in a pH 10 buffer solution as function of time during 60 min
23

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564
of continuous illumination by light from a high power Xe lamp. It was interesting to
observe that the intensity of fluorescence was reduced by 7-8% for the dry state over
the time investigated and with the high flux of photons onto the polymer. However,
under the same conditions, no photobleaching was seen when the polymer was
immersed in a pH 10 buffer solution. The reason for the difference in photostability
between the dry state and wet state is unclear. It could be because the excited single
state of the fluorophore is stabilised by solvation. When compared to the results of
other materials, this still offers excellent performance: the decrease observed in the
fluorescence intensity was 65% for carboxyfluorescein and 10-13%
for
iminocoumarin derivatives, again after 60 min of continuous illumination using a
mercury lamp[7]. Thus an important conclusion is that the material prepared in this
work possesses superior photostability, a feature that is critically important with
excitation of sensor probes by high intensity solid state sources.
in pH 10 buffer solution
1
0.9
dry
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
500
1000 1500 2000 2500 3000 3500 4000
Time (s)
Fig.4. Fluorescence intensity of PVIC (in dry state and in pH 10 buffer solution) as
function of time during 60 min of continuous illumination by light from a high power
24

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581
582
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588
589
Xe lamp (λex = 375 nm, λem = 500 nm). Fluorescence intensities are normalised to the
initial emission of the polymer for each data set.
3.5. Fluorescence studies and response of polymers to pH
Fluorescence measurements of the polymers were performed in 50 mM phosphate
buffer at various pHs as used for the free dyes. The cuvette was shaken vigorously
before each measurement to enable the uniform dispersion of the polymer. No
significant settling out was observed on the timescale of the measurements and the
results were found to be reproducible.
It should be noted that incubation was not required before the measurements as the
response time of the material is very fast (within 10s). This was confirmed by
comparing the data obtained from immediate measurements with those obtained for
the samples that were allowed to incubate for a couple of hours before measurements.
There was essentially no difference between two sets of data.
Fig. 5 shows emission spectra of different polymers at different pHs. It can be seen
that the fluorophores emit at lower wavelengths in the polymers than in their free
forms. The blue shift could be because when fixed in a rigid polymer network the
excited states of the fluorophores undergo less stabilization from solvent
rearrangement.
All polymers exhibited a decrease in fluorescence intensity with increasing pH in the
range from 10 to 13.6, which is similar to the dynamic response range of the free
dyes. The pKa value calculation for the polymers was performed using the Boltzmann
model – Equation (4) where dpH is the slope of the curve within its linear zone.
a ) / dpH
Fmax + Fmin × e( pH − pK
F =
a ) / dpH
e( pH − pK
+1
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598
599
600
601
602
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607
608
609
610
611
612
613
614
(4)
pKa values were found to be 12.65 ± 0.12 , 12.75 ± 0.10 and 12.76 ± 0.14 for PVIC,
PAIC and PSIC, respectively. These values for the immobilized forms of the dyes are
slightly higher than that for their free forms in solution and this arises probably
because of the change in the polarity of the microenvironment.
1.E+06
1.6E+06
1.E+06
a)
1.E+06
1.4E+06
pKa = 12.65 ± 0.12
8.E+05
Increasing pH
1.2E+06
6.E+05
4.E+05
1.0E+06
10 10.5 11 11.5 12 12.5 13 13.5 14
pH
8.0E+05
6.0E+05
4.0E+05
2.0E+05
0.0E+00
400 420 440 460 480 500 520 540 560 580 600
Wavelength (nm)
1.E+06
1.E+06
1.2E+06
1.0E+06
8.0E+05
6.0E+05
4.0E+05
2.0E+05
0.0E+00
b)
8.E+05
6.E+05
4.E+05
2.E+05
0.E+00
pKa = 12.75 ± 0.10
Increasing pH
10 10.5 11 11.5 12 12.5 13 13.5 14
pH
26
350 370 390 410 430 450 470 490 510 530 550
Wavelength (nm)
1.E+06
1.4E+06

615
616
617
618
619
620
621
622
623
624
625
626
627
Fig 5. Emission spectra of PVIC (a), PAIC (b) and PSIC (c) at pH from 10.0 to 13.6.
The insets show the titration plots at emission maxima .
and c).
λex = 370 nm (a), 330 nm (b
628
3.6. Effect of ionic strength (IS)
629
630
631
632
633
634
635
636
637
Sensitivity to IS can be a serious problem in the cases of pH sensors as it affects pKa
values, thus resulting in errors in pH determination. The effect of IS was investigated
with one of the polymers PVIC in the prepared pH 10 phosphate buffer solution
adjusted with NaCl to different ionic strengths ranging from 10 mM to 2000 mM. The
fluorescence intensity obtained for each solution was converted to a pH value using
the calibration curve and the error was calculated. As can be seen from Fig.6, there
appears to be no sensitivity to IS for the sensing polymer at pH 10, even at very high
concentrations of NaCl. The insignificant errors caused are probably due to the
system error rather than the change in IS.
638
27

639
640
641
642
643
644
645
646
647
Fig 6. Effect of ionic strength (IS) on fluorescence intensity and calculated pH value.
Fluorescence intensities are normalised to the maximum emission at 470 nm of the
polymer at pH 10 with IS = 0.
648
649
650
651
652
653
654
655
656
657
658
659
660
4. Conclusions
Novel coumarin-based fluorescent pH indicators vinylbenzylamino imidazolylmethyl
coumarin (VIC), acrylamido imidazolylmethyl coumarin (AIC) and styryl
imidazolylmethyl coumarin (SIC) have been synthesized. Their fluorescence
behaviours have been investigated in aqueous solution at different pHs. All coumarins
exhibited a significant decrease in fluorescence intensity with increasing pH in the
highly alkaline region. Photophysical properties of the indicators have been postulated
to explain their fluorescence properties and behaviours.
The indicators were covalently immobilized to polymer supports by co-polymerising
with methacrylic acid co-monomer and 1,4-bis(acryloyl)piperrazine cross-linker.
These polymers are sensitive to pH in the similar response range of the free dyes and
28