Solid fluorescence sensors obtained by functionalization of photocrosslinked water-swollen acrylic membranes with 4-piperazine naphthalimide derivatives

Article abstract of DOI:10.1016/j.polymer.2017.07.039

A photocrosslinked membrane (M-Cl) based on vinyl-pyrrolidone/butyl acrylate containing ethylene glycol dimethacrylate as a crosslinking agent and methacryloyl chloride as reactive monomer has been prepared for further functionalization. A series of hydroxy substituted piperazine naphthalimide compounds have been synthetized as pH sensitive Off-On fluorescence probes using two methods: microwave and reflux. Three of the derivatives were selected to be anchored to the M-Cl membrane through the acid chloride groups by the Schotten-Baumann reaction. All the obtained water-swollen membranes were characterized using different techniques. Photo-induced electron transfer (PET) and its pH-dependent optical changes were investigated and various photochemical parameters were determined by using pH-dependent absorption and fluorescence spectroscopies. In the pH range of 9.0–4.0, these solid sensors undergo PET process from the piperazine to the naphthalimide moiety leading to a fluorescence quenching. However, in the pH range of 4.0–1.0, the PET is inhibited to give a fluorescence enhancement. The sensitivity of the functionalized membranes to pH changes depended on the size and position of the naphthalimide substituents.

Full text of DOI:10.1016/j.polymer.2017.07.039

Accepted Manuscript
Solid fluorescence sensors obtained by functionalization of photocrosslinked water-
swollen acrylic membranes with 4-piperazine naphthalimide derivatives
S. Fernández-Alonso, T. Corrales, J.L. Pablos, F. Catalina
PII:
S0032-3861(17)30706-1
10.1016/j.polymer.2017.07.039
JPOL 19851
DOI:
Reference:
To appear in: Polymer
Received Date: 20 April 2017
Revised Date: 10 July 2017
Accepted Date: 14 July 2017
Please cite this article as: Fernández-Alonso S, Corrales T, Pablos JL, Catalina F, Solid fluorescence
sensors obtained by functionalization of photocrosslinked water-swollen acrylic membranes with 4-
piperazine naphthalimide derivatives, Polymer (2017), doi: 10.1016/j.polymer.2017.07.039.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
13
11
9
u
7
5
3
1
M-4a
2
4
6
10
12
pH8

ACCEPTED MANUSCRIPT
Solid fluorescence sensors obtained by functionalization of
photocrosslinked water-swollen acrylic membranes with 4-piperazine
naphthalimide derivatives.
S. Fernández-Alonso, T. Corrales, J.L. Pablos, F. Catalina*.
Departamento de Química Macromolecular Aplicada, Instituto de Ciencia y Tecnología
de Polímeros (CSIC). C /Juan de la Cierva, 3. 28006-Madrid, Spain.
E-mail: fcatalina@ictp.csic.es
Abstract
A photocrosslinked membrane (M-Cl) based on vinyl-pyrrolidone/butyl acrylate
containing ethylene glycol dimethacrylate as a crosslinking agent and methacryloyl
chloride as reactive monomer has been prepared for further functionalization. A series
of hydroxy substituted piperazine naphthalimide compounds have been synthetized as
pH sensitive Off-On fluorescence probes using two methods: microwave and reflux.
Three of the derivatives were selected to be anchored to the M-Cl membrane through
the acid chloride groups by the Schotten-Baumann reaction. All the obtained water-
swollen membranes were characterized using different techniques. Photo-induced
electron transfer (PET) and its pH-dependent optical changes were investigated and
various photochemical parameters were determined by using pH-dependent absorption
and fluorescence spectroscopies. In the pH range of 9.0-4.0, these solid sensors undergo
PET process from the piperazine to the naphthalimide moiety leading to a fluorescence
quenching. However, in the pH range of 4.0–1.0, the PET is inhibited to give a
fluorescence enhancement. The sensitivity of the functionalized membranes to pH
changes depended on the size and position of the naphthalimide substituents.
Keywords: photopolymerization, membrane functionalization, naphthalimide
fluorescence probe.
1. Introduction
Polymeric membranes have been widely utilized as a support material for various types
of sensors and also used in systems where the environmental information is gathered by
the measurement of photons. The most commonly used method is fluorescence
emission. Its applications include optical chemical [1,2] and biological [3] sensors.
Hydrophilic polymer matrices are widely used for immobilization of optical indicators
1

ACCEPTED MANUSCRIPT
due to their advantages such as fast response time, low cost, flexibility and their
capability for deposition into various types of substrates.
In recent years, the developments of fluorescence pH sensors have attracted
considerable attention because of their potential application in environmental analytics,
medical diagnostics and process control [4,5]. Compared to small organic compounds,
polymer based optical sensors display several important advantages as they can be non-
invasive or minimally invasive, disposable, easily miniaturized and simple to process as
a membrane, coating or solid layer on adequate surfaces [6,7]. The most widely used
technique amongst optical sensors is the measurement of fluorescence intensity. Among
the large number of fluorescent structures that have been developed, 1,8-naphthalimide
derivatives have numerous applications in a variety of different areas. They have gained
increasing interest as fluorescent probes for pH [8,9], metal cations [10,11,12,13]
and anions [ 14 ] because they have been recognized for excellent photophysical
properties with excellent stability, visible excitation and emission exhibiting high
fluorescence quantum yields and large Stokes' shift that minimize the effects of the
background fluorescence.
Polymerizable naphthalimide derivatives have been copolymerized with different co-
monomers such as methyl methacrylate [15] and styrene [16,17], 2-hydroxyethyl
methacrylate [18] and acrylamide [19,20]. In particular alkylpiperazinyl derivatives of
naphthalimides have been studied and their behavior as proton “off–on” switch sensors
has been demonstrated as very sensitive [21,22] and interesting for applications [23] in
complex media.
In an earlier work, we modified the surface of a film based on a copolymer of ethylene
butyl acrylate with a naphthalimide derivative as an acidity sensor [24]. Even though
the functionalized film was effective as an acid sensor, the hydrophobicity of the
polyolefin surface modified with 4-dimethylamino-1,8-naphthalimide (water contact
angle value of 92.6º) increased the time of its response to acid media.
Vinyl-pyrrolidone (VP) based materials exhibit highly interesting properties due to its
unique combination of physical and chemical properties (biocompatibility, nontoxicity,
chemical stability, good solubility in water and many organic solvents, affinity to both
hydrophobic and hydrophilic complex substances). Also, VP copolymers with acrylic
monomers such as butyl acrylate (BA) have important applications [25,26].
2

ACCEPTED MANUSCRIPT
In this work we have prepared a photocrosslinked membrane based in vinyl-
pyrrolidone/ butyl acrylate and added ethylene glycol dimethacrylate as the crosslinking
agent and methacryloyl chloride as the reactive monomer for further functionalization.
Through the acid chloride groups, three derivatives of naphthalimide were anchored to
obtain solid fluorescence sensors. The membrane preparation and the functionalization
with naphthalimides are shown in Fig. 1.
Fig.1. Synthesis of the photocrosslinked acrylic membrane containing acid chloride
groups (M-Cl) and functionalization with the selected naphthalimide structures.
The naphthalimides linked to the membrane containing acid chloride (M-Cl) were
selected from a series of naphthalimide derivatives previously synthetized in this work
using two methods: microwave and reflux. The structures of the series (Fig. 2) are
hydroxyl derivatives of piperazine 1,8-naphthalimides which have been reported to have
excellent fluorescence properties [21,22]. The membranes were characterized and the
spectroscopic characteristics of the naphthalimide derivatives and the membranes were
3

ACCEPTED MANUSCRIPT
studied in function of pH and of different solvent polarities using UV-vis and
fluorescence spectroscopies.
2. Experimental
2.1. Materials and reagents
All materials and solvents were commercially available and used as received, unless
otherwise indicated. The materials included: sodium hydroxide (Panreac, 98-100.5%),
hydrochloric acid (VWR Chemicals, 37%), ultrapure MilliQ water (Millipore),
triethylamine (Aldrich, ≥99%), 4-bromo-1,8-naphthalic anhydride (Aldrich, 95%),
potassium carbonate (Panreac, 99%), 2-aminoethanol (Aldrich, 99%), 2-
methoxyethylamine (Aldrich, 99%), piperazine (Aldrich, 99%), 1-methylpiperazine
(Aldrich,
99%),
1-(2-hydroxyethyl)piperazine
(Aldrich,
98%),
1-[2-(2-
hydroxyethoxy)ethyl]piperazine (Aldrich, 95%) ethanol (VWR Chemicals, 99.95%),
dimethylformamide (Scharlau, 99.8%), Irgacure 2959 photoinitiator (BASF), ethyl
acetate (Aldrich, ≥99.5%), hexane (Carlo Erba Reagents, 99%), 2-propanol (Scharlau,
99.5%), dichloromethane (Aldrich, ≥99.9%), toluene (Merck, ≥99.9%), diethyl ether
(Carlo Ebra, >99.8%), 1,4-dioxan (Panreac, 99%), tetrahydrofuran (Aldrich, 99.9%),
chloroform (Scharlau, 99%), acetone (Scharlau, 99.5%), acetonitrile (Aldrich, ≥99.5%),
1-butanol (Panreac, 99.5%), methanol (Aldrich, ≥99.8%), coumarin 6 (Aldrich, 98%),
dimethylsulfoxide D6 (Euriso-top, 99.8% D), chloroform-d (Aldrich, 99.8% D),
diiodomethane (Aldrich, 99%).
Monomers, 1-vinyl-2-pyrrolidone (Aldrich, ≥97%), butyl acrylate (Aldrich, ≥99%) and
ethylene glycol dimethacrylate (Aldrich, 98%) and methacryloyl chloride (Aldrich,
≥97%) were distilled under vacuum to remove the inhibitor before use.
2.2. Spectroscopic characterization and thermal analysis
Attenuated Total Reflectance / FT-Infrared Spectroscopy (ATR-FTIR) was used to
characterize naphthalimide derivatives and membranes. ATR-FTIR spectra were
obtained using a PERKIN ELMER BX-FTIR Spectrometer coupled with a
MIRacleTMATR accessory, from PIKE Technologies and interferograms were obtained
from 32 scans.
4

ACCEPTED MANUSCRIPT
UV spectroscopy was employed for the quantitative determination of each hydroxyl-
piperazine-naphthalimide derivative anchored to acrylic membranes in a PERKIN
ELMER Lambda 35 spectrometer. The naphthalimide content was determined through
measurement of absorbance at the peak maximum of the absorption band. Assessment
was made in quintuplicate for each material.
Fluorescence spectra were recorded using a Perkin Elmer LS 55 Fluorescence
Spectrometer. Fluorescence emission spectra of the probe were recorded in the range
490–700 nm using as the excitation wavelength the maximum of the longest wavelength
absorption band. All the spectra were corrected using the response curve of the
photomultiplier. The fluorescence quantum yields (φF) were measured relatively to
Coumarin 6 (φF =0.78 in ethanol) [27].
1
13
Nuclear magnetic resonance, H- and C- NMR spectra were recorded on a Varian-
Mercury 400 MHz and a Bruker 300 MHz Nuclear Magnetic Resonance Spectrometers
using hexadeuterated dimethyl sulfoxide (DMSO-d6) and chloroform-d (CDCl3) as the
1
13
solvent. Chemical shifts were reported in parts per million (ppm). H-NMR and C-
NMR chemical shifts were referenced to DMSO-d6 (2.5 and 39.52 ppm respectively) or
CDCl3 (7.25 and 77.0 ppm respectively) as standard.
Mass Spectra (MS) were recorded on a HP 5973-MSD spectrometer.
Differential Scanning Calorimetry (DSC) was performed on a METTLER DSC-823e
instrument (30–180◦C) previously calibrated with an indium standard (Tm= 429 K,
1
∆Hm= 25.75 Jg− ). Samples (10 mg) were tightly sealed onto the DSC pans and heating
or cooling at 10◦C·min-1 rates under nitrogen. DSC was used to measure the glass
transition temperature of the acrylic membranes and the melting points of the
naphthalimide derivatives. The glass transition temperature of the membranes (Tg) were
determined in a second scan after erasing the previous thermal history of the material in
a first scan. rate of 10 ◦C/min under nitrogen flow (30 psi).
Thermogravimetric Analysis (TGA) was carried out in a TGA Q-500 TA Instruments
coupled to a Pfeiffer Vacuum ThermoStarTM mass spectrometer. The weight loss was
measured as a function of temperature, and the evolved gas masses were directly
monitored. The heating rate for the dynamic conditions was 10 ºC·min-1, and the
nitrogen flow was maintained constant at 60 ml·min-1. The onset degradation
temperature (TOnset) was defined as the initial temperature of degradation, corresponding
5

ACCEPTED MANUSCRIPT
to the intersection of the tangent drawn at the decomposition step with the horizontal
zero-line of the TG curve.
Elemental analysis (EA) was carried out on a Carlo Erba EA1108 elemental analyzer (%
of C, H, N).
2.3. Swelling Ratio and water contact angle measurements
Swelling degree (SD) in water and THF of the crosslinked membranes were measured
by immersing dried and weighed sample films (Wd) into deionized water or THF at
30ºC for 24 h. The superficial solvent was removed and the samples were immediately
weighed (Ws). The SD were calculated as follows: SD (%) = ((Ws −Wd)/Wd)×100,
where Ws and Wd are the weight of the swollen and dry sample, respectively.
Contact angle (CA) measurements were performed at 25 °C using a KSV instruments
LTD CAM200 Tensiometer and MilliQwater as wetting solvent. Surface energy was
determined using two liquids (water and methylene iodide) for the measurements. On
the basis of Owens–Wendt's method [28], the surface energy (γ) and its dispersive
(γd) and polar (γp) parts were calculated using the CAM 200 software.
2.4. Microwave and UV irradiation equipments
The microwave equipment used in this work was an Anton Paar MonowaveTM 300
microwave synthesis reactor provided with an infrared sensor (IR pyrometer). All
reactions were performed in pressure-resistant 30-mL test tubes sealed with silicon
septum and using a magnetic stirring bar.
The photopolymerization reactions were carried out in a Biolink™ BLX-365 type Bio-
link apparatus (Vilbert Lourmat™).
2.5. Membrane preparation by photopolymerization (M-Cl) and functionalization with
HO-piperazine-naphthalimide derivatives
The reaction scheme for the photopolymerization reaction and the membrane
functionalization with naphthalimide is shown in Figure 1.
6

ACCEPTED MANUSCRIPT
A crosslinked membrane (M-Cl) was obtained by the bulk photopolymerization of a
mixture of monomers: N-vinylpyrrolidone (VP), butyl acrylate (BA), ethylene glycol
dimethacrylate (EGDMMA) as the crosslinking agent and methacryloyl chloride
(MACl) as the reactive monomer. The monomer molar ratio of the feed mixture was
VP:55/BA:35/ MACl:10/EGDMMA:5 and the photoinitiator (Irgacure 2959) was added
to the mixture at 1% (w/w). The homogenous mixture was transferred to an ampoule,
degassed by argon bubbling for 15 min and injected into a previously degassed silanized
glass mould. The two glass pieces were separated by a Teflon spacer of 180 µm thick to
define the reaction chamber. After 40 minutes of irradiation at 365 nm (dose 20 J cm−2),
the transparent crosslinked membrane (180 µm of thickness) was demoulded and
conditioned (12h) at room temperature under argon atmosphere. The crosslinked
membranes were then washed several times with ethanol and dried under vacuum at 40
°C to remove the un-reacted monomers. The presence of acid chloride was confirmed
by ATR-FTIR (wavenumbers,cm−1), νC=O acyl chloride 1795 cm-1; νC-Cl 898 cm-1
together with the characteristic co-monomers bands of VP νC=O lactam, amide-I 1675
cm-1 and BA, νC=O ester 1725 cm-1; νC-O (O-CH2-) ester 1161 cm-1. The quantitative
determination of chlorine in the membrane (2.8%) was carried out by TGA-MS (Table 1
and Fig. 3B and C).
In a second step, the M-Cl film was cut into strips (1 × 4 cm) and functionalized in the
solid state with the naphthalimide derivatives synthetized in this work (Fig 2). The
procedure was as follows: the M-Cl membrane was placed in an ace round-bottom
pressure flask with 20 mL of THF. 10 mg of hydroxy-piperazine naphthalimide
derivative (4a, 5b or 6b) and 500 µL of triethylamine were added under argon
atmosphere. The mixture was heated at 55°C for 15h. After this reaction time, the
corresponding modified membranes M-4a, M-5b and M-6b were washed with cold
ethanol and with water thrice in order to remove the unreacted acid chloride groups and
the unreacted naphthalimide. The hydrolysis reaction of unreacted acid chloride groups
was confirmed by ATR-FTIR and by TGA-MS as the characteristic peak of acid
chloride was not present (Fig. 3C). After drying, the transparent functionalized
membranes exhibited an intense yellow–green fluorescence and the content of
naphthalimide was quantitatively determined by UV–Vis spectroscopy (Table 1). The
characteristic peaks of naphthalimide structures in the membranes were not clearly
7

ACCEPTED MANUSCRIPT
observed in the ATR-FTIR spectra due to their low intensity and their overlap with the
broad absorptions of the membrane functional groups.
2.6. Synthesis of the naphthalimide derivatives
Compound 2a was synthesized following the procedure (Fig. 2) described in the
bibliography All the naphthalimide derivatives were synthetized using microwave
(MW) and reflux (RF) methods with the same experimental conditions but differing in
reaction times and yields. The RF reactions times were larger (15 h for all the reactions)
and RF yields were lower than those obtained under MW irradiation. The synthesis
path-ways, naphthalimide structures and reaction data are shown in Fig.2.
MW method
Reflux method
Structure
t (h.)
Y (%)
t (h.)
Y (%)
2a
2b
3a
4a
5a
6a
3b
4b
5b
6b
2
2
1
1
1
1
1
1
1
1
90
93
84
85
88
85
84
83
84
85
15
15
15
15
15
24
15
15
15
24
76
80
64
67
68
70
69
73
71
69
8

ACCEPTED MANUSCRIPT
Fig.2. Synthesis of the naphthalimide derivatives and their corresponding reaction times
and yields for microwave (MW) and reflux (RF) methods.
To synthesize the desired piperazinyl derivative of naphthalimide, two bromide
derivatives of naphthalimide containing 2-hydroxyethyl (Compound 2a) and 2-
methoxyethyl (Compound 2b) groups were synthesized following the procedure
described in the bibliography [ 29 , 30 ]. In a second step, the bromide atom was
substituted by piperazinyl groups (Fig. 2) following a modified method similar to that
reported by Tian et al. [19].
2.6.1. Synthesis of N-(2-hydroxyethyl)-4-bromo-1,8-naphthalimide (2a)
In a pressure-resistant microwave test tube, a mixture of 4-bromo-1,8-naphthalic
anhydride (compound 1) (1.4 g, 5 mmol) and ethanol amine (0.4 g, 5 mmol) in ethanol
(15 ml) was heated under argon at 85 ºC and stirred at 600 rpm for 2h. The resulting
mixture was cooled at 5 °C. The solid separated was filtered and washed with cold
ethanol (3 x 30 ml) and after dried was identified as N-hydroxyethyl-4-bromo-1,8-
1
naphthalimide (compound 2a). Yield 90% (1.4 g). M.p.: 206 ± 2ºC. H-NMR (δH ppm)
(300 MHz, DMSO-d6, Me4Si): δ 8.42 (dd, J = 13.9, 7.9 Hz, 2H), 8.20 (d, J = 7.9 Hz,
1H), 8.10 (d, J = 7.8 Hz, 1H), 7.89 (t, J = 7.9 Hz, 1H), 4.81 (s, 1H), 4.10 (t, J = 6.4 Hz,
13
2H), 3.61 (t, J = 5.9 Hz, 2H). C-NMR (δC ppm) (100.6 MHz, DMSO-d6, Me4Si): δ
162.90, 162.85, 132.41, 131.42, 131.24, 130.80, 129.61, 128.98, 128.67, 128.14,
122.70, 121.92, 57.71, 41.95. FT-IR (wavenumbers, cm-1): νOH 3386 cm-1; νC-H 3066
cm-1 aromatic stretch vibration νC=O 1692, 1658 cm-1; νN-C=O 1611 cm-1; νC-C 1585, 1568
cm-1 aromatic ring chain vibrations. EA: theoretical values: %C 52.52; %H 3.15; %N
4.38; experimental values: %C 52.42; %H 3.25; %N 4.25. EI-MS m/z calculated for
C14H10BrNO3 (M+H)+ 321.0; found 321.0.
2.6.2. Synthesis of N-(2-methoxyethyl)-4-bromo-1,8-naphthalimide (2b)
Compound 2b was synthetized following the same procedure described for compound
2a from but using 2-Methoxyethylamine (0.38 g, 5 mmol) as a reactive amine. Yield
9

ACCEPTED MANUSCRIPT
1
93% (1.55 g). M.p.: 163.57 ± 2ºC. H-NMR (δH ppm) (300 MHz, CDCl3, Me4Si): δ
8.62 (d, J = 7.3 Hz, 1H), 8.52 (d, J = 8.4 Hz, 1H), 8.38 (d, J = 7.9 Hz, 1H), 8.00 (d, J =
7.9 Hz, 1H), 7.85 – 7.77 (m, 1H), 4.42 (t, J = 5.8 Hz, 2H), 3.72 (t, J = 5.8 Hz, 2H), 3.37
13
(s, 3H). C-NMR (δC ppm) (100.6 MHz, CDCl3, Me4Si): δ 163.80, 163.78, 133.38,
132.26, 131.43, 131.18, 130.68, 130.40, 129.12, 128.16, 123.09, 122.23, 69.68, 58.92,
39.50. FT-IR (wavenumbers, cm-1): νC-H 3065 cm-1 aromatic stretch vibration; νO-CH3
2824,2832 cm-1; νC=O 1698, 1655 cm-1; νN-C=O 1613 cm-1; νC-C 1589, 1570 cm-1 aromatic
ring chain vibrations. EA: theoretical values: %C 53.91; %H 3.62; % N 4.19;
experimental values % C 53.82; % H 3.65; % N 4.22. EI-MS m/z calculated for
C15H12BrNO3 (M+H)+ 335.0; found 335.0.
Synthesis of compounds 2a and 2b were also carried out in an ace round-bottom
pressure flask and conventional heating during 15 h yielded 75 and 80 % respectively.
2.6.3. Synthesis of N-(2-hydroxyethyl)-4-(piperazine-1-yl)-1,8-naphthalimide (3a)
In a pressure-resistant microwave test tube, compound 2a (0.20 g, 0.625 mmol) and
piperazine (0.16 g, 1.875 mmol) were mixed in 2-methoxyethanol (5 mL) and heated at
130 ºC in argon atmosphere under constant stirring (600 rpm) for 1 h. The mixture was
poured into cooled ice/water at 5 °C and the residue was filtered and washed with hot
ethanol (30 mL). The solution was filtered and the solvent removed under reduced
pressure in order to obtain the yellow solid compound. M.p.: 219.23 ± 2ºC. MW-yield:
84%. RF method for compound 3a yielded a 64% after 15 h of reaction time. 1H NMR
(300 MHz, DMSO-d6) δ 8.48 – 8.41 (m, 2H), 8.38 (d, J = 8.1 Hz, 1H), 7.79 (t, J = 7.8
Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 4.78 (s, 1H), 4.13 (t, J = 6.4 Hz, 2H), 3.60 (s, 2H),
3.13 (s, 4H), 3.00 (s, 4H). 13C NMR (75 MHz, DMSO) δ 163.65, 163.11, 156.20,
132.14, 130.48, 129.15, 125.78, 125.22, 122.59, 115.33, 114.79, 57.86, 54.10, 45.67,
41.60. FT-IR (wavenumbers, cm-1): νNH 3248 cm-1; νC-H 3066 cm-1 aromatic stretch
vibration; νC=O 1685, 1642 cm-1; νN-C=O 1613 cm-1; νC-C 1586, 1571 cm-1 aromatic ring
chain vibrations; νNH 1514 cm-1 flexion. EA: theoretical values: %C 66.45; %H 5.89;
%N 12.91; experimental values: %C 66.55; %H 5.93; %N 12.96. EI-MS m/z calculated
for C18H19N3O3: (M+H)+ 326.1, found. 326.0 (M+).
10

ACCEPTED MANUSCRIPT
2.6.4. Synthesis of N-(2-hydroxyethyl)-4-(4-methylpiperazine-1-yl)-1,8-naphthalimide
(4a)
The synthesis of compound 4a was similar to the preparation procedure of the
compound 3a but using 1-methylpiperazine (208 µL, 1.875 mmol) as a reactive amine.
The MW yield was 85% after 1 h and RF yield 67% after 15h. Compound 4a was
purified by silica gel column chromatography using dichloromethane:methanol (20:1)
1
as an eluent to obtain a yellow solid. M.p.: 188.43 ± 2ºC. Yield: 85%. H NMR (300
MHz, DMSO-d6) δ 8.43 (dd, J = 11.9, 8.1 Hz, 2H), 8.37 (d, J = 8.2 Hz, 1H), 7.79 (t, J =
7.8 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 4.81 (t, J = 5.7 Hz, 1H), 4.12 (t, J = 6.4 Hz, 2H),
3.59 (d, J = 6.2 Hz, 2H), 3.22 (s, 4H), 2.63 (s, 4H), 2.30 (s, 3H).13C NMR (75 MHz,
DMSO) δ 163.61, 163.07, 155.51, 132.04, 130.48, 130.31, 129.08, 125.87, 125.20,
122.59, 115.56, 114.93, 57.85, 54.63, 52.50, 45.74, 41.60. FT-IR (wavenumbers, cm-1):
νOH 3435 cm-1; νC-H 3060 cm-1 aromatic stretch vibration; νC=O 1692, 1635 cm-1; νN-C=O
1612 cm-1; νC-C 1585 cm-1 aromatic ring chain vibrations. Elemental analysis,
theoretical value %C 67.24; %H 6.24; %N 12.38; calculate %C 67.15; %H 6.15; %N
12.29. EI-MS m/z calculated for C19H21N3O3: (M+H)+ 340.2, found. 340.3 (M+).
2.6.5. Synthesis of N-(2-hydroxyethyl)-4-(4-(2-hydroxyethyl)-piperazine-1-yl)-1,8-
naphthalimide (5a)
The synthesis of compound 5a was similar to the preparation procedure of the
compound 3a but using 1-(2-Hydroxyethyl)piperazine (230 µL, 1.875 mmol) as a
reactive amine. Compound 5a was purified by silica gel column chromatography using
dichloromethane:methanol (20:1) as an eluent to obtain an yellow solid. M.p.: 178.36 ±
1
2ºC. The MW yield was 88% after 1 h and RF yield 68% after 15h. H NMR (300
MHz, DMSO-d6) δ 8.49 – 8.41 (m, 2H), 8.39 (d, J = 8.1 Hz, 1H), 7.84 – 7.76 (m, 1H),
7.33 (d, J = 8.1 Hz, 1H), 4.81 (t, J = 5.9 Hz, 1H), 4.49 (t, J = 5.3 Hz, 1H), 4.13 (t, J =
13
6.5 Hz, 2H), 3.65 – 3.51 (m, 6H), 3.23 (s, 4H), 2.74 (s, 4H). C NMR (75 MHz,
DMSO) δ 163.94, 163.41, 155.88, 132.38, 130.82, 130.67, 129.42, 126.19, 125.51,
122.92, 115.85, 115.17, 60.57, 58.95, 58.19, 53.50, 53.00, 41.94. FT-IR (wavenumbers,
cm-1): νOH 3260, 3146 cm-1; νC-H 3052 cm-1 aromatic stretch vibration; νC=O 1695, 1644
cm-1; νN-C=O 1613 cm-1; νC-C 1589, 1576 cm-1 aromatic ring chain vibrations. EA:
theoretical values %C 65.03; %H 6.28; %N 11.37; experimental values: %C 65.00; %H
11

ACCEPTED MANUSCRIPT
6.25; %N 11.31. EI-MS m/z calculated for C20H23N3O4: (M+H)+ 370.17, found 370.2
(M+).
2.6.6. Synthesis of N-(2-hydroxyethyl)-4-(4-[2-(2-hydroxyethoxy)ethyl]-piperazine-1-
yl)-1,8-naphthalimide (6a)
The synthesis of compound 6a was similar to the preparation procedure of the
compound 3a but using 1-[2-(2-hydroxyethoxy)ethyl]piperazine (308 µL, 1.875 mmol)
as a reactive amine. After completion of the reaction, the solvent was evaporated under
reduced pressure and the crude product was then diluted in 50 ml of water. The
precipitated product was then filtered off. The aqueous part was extracted with
dichloromethane (3 x 30 mL) and the organic layer was dried over K2CO3. The solvent
was evaporated under reduced pressure to obtain a yellow solid. M.p.: 170.20± 2ºC. For
1
compound 6a the MW yield was 85% after 1 h and RF yield 70% after 24h. H NMR
(300 MHz, DMSO-d6) δ 8.47 – 8.39 (m, 2H), 8.37 (d, J = 8.1 Hz, 1H), 7.79 (t, J = 7.9
Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 4.79 (t, J = 5.8 Hz, 1H), 4.63 (t, J = 5.0 Hz, 1H), 4.13
(t, J = 6.5 Hz, 2H), 3.62 – 3.54 (m, 4H), 3.54 – 3.48 (m, 2H), 3.45 (d, J = 4.6 Hz, 2H),
3.22 (s, 4H), 2.75 (s, 4H), 2.65 – 2.58 (m, 2H).13C NMR (75 MHz, DMSO) δ 163.63,
163.09, 155.52, 132.07, 130.51, 130.35, 129.08, 125.88, 125.18, 122.58, 115.53,
114.88, 72.29, 68.31, 60.32, 57.88, 57.28, 53.15, 52.64, 41.62. FT-IR (wavenumbers,
cm-1): νOH 3374 cm-1; νC-H 3066 cm-1 aromatic stretch vibration; νC=O 1690, 1650 cm-1;
νN-C=O 1616 cm-1; νC-C 1588 cm-1 aromatic ring chain vibrations. EA: theoretical values
%C 63.91; %H 6.58; %N 10.16; experimental values: %C 63.97; %H 6.62; %N 9.60.
EI-MS m/z calculated for C22H27N3O5: (M+H)+ 414.20, found 414.2 (M+).
2.6.7. Synthesis of N-(2-methoxyethyl)-4-(piperazine-1-yl)-1,8-naphthalimide (3b)
Compound 2b (0.20 g, 0.598 mmol) and piperazine (0.155 g, 1.794 mmol) were mixed
in 2-methoxyethanol (5 mL) and heated under MW radiation at 130 ºC in an argon
atmosphere under constant stirring (600 rpm) for 1 h. After completion of the reaction,
the solvent was evaporated under reduced pressure and the crude was washed with
water and filtered to obtain the compound as a yellow solid. M.p.: 145.60 ± 2º The MW
yield was 84% after 1 h and by conventional heating, RF yield 69% after 15h. 1H NMR
(300 MHz, DMSO-d6) δ 8.48 – 8.41 (m, 2H), 8.40 (s, 1H), 7.83 – 7.75 (m, 1H), 7.30 (d,
12

ACCEPTED MANUSCRIPT
J = 8.1 Hz, 1H), 4.23 (t, J = 6.1 Hz, 2H), 3.57 (t, J = 6.1 Hz, 2H), 3.26 (s, 3H), 3.15 (s,
4H), 3.00 (s, 4H). 13C NMR (75 MHz, DMSO) δ 163.50, 162.94, 156.29, 132.25,
130.58, 129.09, 125.75, 125.17, 122.33, 115.00, 114.77, 68.67, 57.94, 54.08, 45.66,
38.33. FT-IR (wavenumbers, cm-1): νNH 3248 cm-1; νC-H 3069 cm-1 aromatic stretch
vibration; νO-CH3 2828, 2808 cm-1 νC=O 1690, 1650 cm-1; νN-C=O 1613 cm-1; νC-C 1588,
1572 cm-1 aromatic ring chain vibrations; νNH 1514 cm-1 flexion. EA: theoretical values
%C 67.24; %H 6.24; %N 12.38; experimental values: %C 67.20; %H 6.18; %N 12.31.
EI-MS m/z calculated for C19H21N3O3: (M+H)+ 340.1, found. 340.0 (M+).
2.6.8. Synthesis of N-(2-methoxyethyl)-4-(4-methylpiperazine-1-yl)-1,8-naphthalimide
(4b)
Compound 4b was synthetized by mixing compound 2b (0.2 g, 0.598 mmol), 1-
methylpiperazine (200 µL, 1.7964 mmol) in 5 mL of 2-methoxyethanol and following
the same procedure as for compound 3b. M.p.: 139.44 ± 2ºC. The MW yield was 83%
after 1 h and by conventional heating, RF yield 73% after 15h. 1H NMR (300 MHz,
DMSO-d6) δ 8.42 (dd, J = 12.3, 8.0 Hz, 2H), 8.36 (d, J = 8.1 Hz, 1H), 7.83 – 7.73 (m,
1H), 7.31 (d, J = 8.2 Hz, 1H), 4.22 (t, J = 6.1 Hz, 2H), 3.57 (t, J = 6.1 Hz, 2H), 3.25 (s,
13
3H), 3.22 (s, 4H), 2.63 (s, 4H), 2.30 (s, 3H). C NMR (75 MHz, DMSO) δ 163.51,
162.97, 155.66, 132.21, 130.64, 130.51, 129.08, 125.93, 125.21, 122.40, 115.29,
114.99, 68.67, 57.94, 52.49, 45.73, 38.36. FT-IR (wavenumbers, cm-1): νC-H 3067 cm-1
aromatic stretch vibration; νC=O 1695, 1650 cm-1; νN-C=O 1613 cm-1; νC-C 1586, 1574 cm-1
aromatic ring chain vibrations. EA: theoretical values %C 67.97; %H 6.56; %N 11.89;
experimental values %C 67.87; %H 6.56; %N 11.80. EI-MS m/z calculated for
C20H23N3O3: (M+H)+ 354.17, found 354.2 (M+).
2.6.9. Synthesis of N-(2-methoxyethyl)-4-(4-(2-hydroxyethyl)-piperazine-1-yl)-1,8-
naphthalimide (5b)
The synthesis of compound 5b was similar the preparation procedure of the compound
3b but using 1-(2-Hydroxyethyl)piperazine (220 µL, 1.796 mmol) as a reactive amine.
Compound 5b was purified by silica gel column chromatography using
dichloromethane:methanol (20:1) as an eluent to obtain an yellow solid. M.p.: 124.17 ±
2ºC. Yield: 84%.1H NMR (300 MHz, Chloroform-d) δ 8.57 (d, J = 7.2 Hz, 1H), 8.50
13

ACCEPTED MANUSCRIPT
(d, J = 8.0 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 8.0
Hz, 1H), 4.41 (t, J = 5.6 Hz, 2H), 3.71 (t, J = 5.3 Hz, 4H), 3.36 (s, 3H), 3.30 (s, 4H),
2.84 (s, 4H), 2.75 – 2.65 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 164.94, 164.45, 156.25,
133.07, 131.63, 130.66, 130.32, 126.53, 126.05, 123.55, 117.09, 115.33, 70.09, 59.83,
59.18, 58.31, 53.48, 53.41, 39.49. FT-IR (wavenumbers, cm-1): νOH 3140 cm-1 ; νC-H
3052 cm-1 aromatic stretch vibration; νC=O 1696, 1645 cm-1; νN-C=O 1613 cm-1; νC-C 1589,
1576 cm-1 aromatic ring chain vibrations. Elemental analysis, theoretical value %C
65.78; %H 6.57; %N 10.96; calculate %C 65.87; %H 6.68; %N 11.04. EI-MS: m/z
calculated for C21H25N3O4 (M+H)+ 384.18; found 384.2 (M+).
2.6.10. Synthesis of N-(2-methoxyethyl)-4-(4-[2-(2-Hydroxyethoxy)ethyl]-piperazine-1-
yl)-1,8-naphthalimide (6b)
Compound 2b (0.2 g, 0.5988 mmol), 2-[2-(1-Piperazinyl)ethoxy]ethanol (295µL,
1.7964 mmol) was mixed in 5 mL of 2-methoxyethanol and heated at 130 °C in argon
atmosphere. The mixture was stirred at 600 rpm for 1 h. After completion of the
reaction, the solvent was evaporated under reduced pressure and the crude product was
then diluted in 50 ml of water. The precipitated product was then filtered off. The
aqueous part was extracted with dichloromethane (3 x 30 mL) and the organic layer was
dried over K2CO3. The solvent was evaporated under reduced pressure to obtain a
yellow solid. M.p.: 219.73 ± 2ºC. The MW yield was 85% after 1 h and by conventional
heating, RF yield 69 % after 24 h. 1H NMR (300 MHz, DMSO-d6) δ 8.43 (dd, J = 11.0,
8.0 Hz, 2H), 8.37 (d, J = 8.1 Hz, 1H), 7.82 – 7.75 (m, 1H), 7.31 (d, J = 8.2 Hz, 1H),
4.64 (s, 1H), 4.22 (t, J = 6.1 Hz, 2H), 3.58 (q, J = 6.0 Hz, 4H), 3.53 – 3.48 (m, 2H), 3.45
(d, J = 4.7 Hz, 2H), 3.25 (s, 3H), 3.22 (s, 4H), 2.75 (s, 4H), 2.62 (t, J = 5.8 Hz, 2H).13C
NMR (75 MHz, DMSO) δ 163.54, 162.99, 155.68, 132.24, 130.67, 130.55, 129.08,
125.94, 125.20, 122.39, 115.27, 114.94, 72.28, 68.70, 68.31, 60.30, 57.97, 57.26, 53.13,
52.64, 38.39. FT-IR (wavenumbers, cm-1): νO-H 3356 cm-1; νC-H 3065 cm-1 aromatic
stretch vibration; νC=O 1687, 1648 cm-1; νN-C=O 1613 cm-1; νC-C 1585 cm-1 aromatic ring
chain vibrations. EA: theoretical value %C 64.62; %H 6.84; %N 9.83; experimental
values %C 64.60; %H 6.93; %N 9.76. EI-MS: m/z calculated for C23H29N3O5 (M+H)+
428.2; found 428.3 (M+).
14

ACCEPTED MANUSCRIPT
2.7. pH measurements
Measurements of pH were obtained with a Mettler Toledo SevenGo Duo Pro meter with
an InLab Expert Pro ISM-ID67 electrode at room temperature (23ºC) and previously
calibrated with standard buffers. The sensing measurements determined from the
solutions of naphthalimide derivatives and functionalized membranes were performed
using HCl and NaOH solutions for pH variation in the mixture water:ethanol (4:1) as
solvent.
The acid titration by UV/Vis and fluorescence in aqueous solution was performed as
follows. The titration in solution with naphthalimide derivatives (water:ethanol 4:1,
naphthalimide concentration 10-4 M and pH=12.0 fixed by addition of NaOH solution)
was carried out by increasing the acidity from pH 12.0 to pH 2.0 by adding aliquots of
the diluted hydrochloric acid. After each addition, the pH was measured after the
solutions were allowed to equilibrate for 10 min and the UV/Vis and fluorescence
spectra were recorded.
In the case of functionalized crosslinked membranes, films were cut into strips of 1x4
cm and fixed in a homemade support that can also be slotted into the cell holder of the
spectrophotometers. To study the effect of the pH in the spectroscopic properties of the
membranes, film strips were immersed in previously prepared vials (50 mL) containing
solutions from pH 12.0 to pH 2.0. The UV/Vis and fluorescence spectra for each pH
were taken after 20 min. of conditioning time.
3. Results and discussion
3.1. Synthesis of naphthalimide derivatives
The synthesis route [3,4] to obtain N-hydroxy alkyl piperazine naphthalimides is
presented in Fig. 2. In the first step, the condensation of 4-bromo-1,8-naphthalic
anhydride with 2-hydroxyethylamine (series a) or 2-methoxyethylamine (series b) under
microwave irradiation in ethanol at 85 °C afforded 2a and 2b respectively in high yield
at a shorter reaction time than conventional conditions.
15

ACCEPTED MANUSCRIPT
In the second reaction step (Fig. 2), compounds 2a and 2b reacted with 3 equiv. of each
piperazine derivative as detailed in the experimental description (section 2.6.) to obtain
the piperazine-naphthalimides derivatives shown in Fig. 2. Hence, we have prepared
piperazine substituted derivatives with hydrogen and methyl groups [19] as substituents
on the amine groups (structures 3 and 4 respectively), but also with bigger substituents
(structures 5 and 6) in order to study comparatively their PET processes and their
protonation in acid media.
The best synthesis conditions were noted under MW irradiation for 1 h at 850W and a
ceiling temperature of 130 °C in 2-methoxyethanol. In contrast, the best conditions for
the synthesis carried out under reflux by conventional heating required reaction times of
15 h and 24 h for compounds 6a and 6b when 2-[2-(1-Piperazinyl)ethoxy]ethanol was
used as reactive amine.
In all the cases, MW irradiation allowed higher yields at shorter reaction times than the
conventional RF method. These results are in agreement with those reported in the
literature [24,19].
3.2. Membrane preparation, functionalization with piperazine naphthalimides sensors
and characterization
The membrane prepared containing acid chloride functionalities (M-Cl) as a base
material for further functionalization was obtained by bulk photopolymerization of a
mixture of monomers combining the hydrophilic character of VP (55%) with the
hydrophobic nature of BA (35%) and adding MACl (10%) as the reactive monomer for
functionalization. A 5% of EGDMMA as the crosslinking agent of the mixture
controlled the crosslinking degree of the final membrane. This produced a reversible
water-swollen membrane (Fig. 1). After the bulk photopolymerization, all the
experiments reached >98 % of conversion (determined by weight differences), and the
resulting crosslinked acrylic membrane contained 2.8% of chlorine in the structure (Fig.
3B).
In a second step, the M-Cl membrane was functionalized with the three naphthalimide
derivatives from the series synthetized in this work (Fig. 1) that contained mono
hydroxy alkyl piperazine, derivatives 4a, 5b and 6b. This functionalization was
16

ACCEPTED MANUSCRIPT
accomplished successfully by conventional heating in THF at 55ºC for 15h under very
low stirring speed. This selected reaction temperature is 10°C below the THF boiling
point to be sure there isn't any damage of the membrane in the heterogeneous reaction,
such as breaks, wrinkles and warps that could decrease the optical quality of the
membrane. Under these experimental conditions, the Schotten-Baumann reaction used
to graft the naphthalimide derivatives to the M-Cl membrane proceeded very slowly.
This allowed a good control in homogeneity of functionalization without physically
damaging the membrane. The unreacted acid chloride group disappeared after
hydrolysis when the membranes were washed as shown in Fig 3C. The amount of
naphthalimide grafted to the membranes (Table 1) was determined from the UV-Vis
absorbance using the absorption coefficient of each derivative (Table 3) previously
calculated in water-ethanol (4:1, v/v).
Table 1. Content of chlorine and naphthalimide derivative in the membranes and
thermal properties determined by DSC and TGA.
Cl
Content(a)
(%)
Naphthalimide
Content(b)
(c)
(d)
(d)
Tg
T5
T10
Membrane
(ºC)
(°C)
(°C)
(mol·L-1) x103
M-Cl
2.8
0
0
-
46.9
45.3
45.4
45.6
214
326
337
334
337
369
371
372
M-4a
M-5b
M-6b
2.73
3.27
4.72
0
a Determined by TGA peak at 175ºC corresponding to Cl, EI-MS m/z 36 (M+); b Determined from
the membrane (180 microns of thickness) by UV spectroscopy; c Measured by DSC second heating
run at a heating rate of 10ºC·min-1; d Temperatures corresponding to 5% and 10% weight loss.
After 15 hours of reaction time the content of the naphthalimide derivative in the
membrane was very low (Table 1) but the concentration is adequate for sensing
applications by UV-vis and fluorescence spectroscopies.
The good physical characteristics of the functionalized membranes are similar than
those observed by other authors [25] with copolymers of VP/BA. All DSC thermograms
of the membranes revealed the presence of a single glass transition temperature for all
the membranes confirming copolymer miscibility. The characteristic Tg values for the
membranes are shown in Table 1 and the DSC thermograms (second scan) obtained for
17

ACCEPTED MANUSCRIPT
M-Cl and M-5b samples are plotted in Fig. 3A. The observed step for M-Cl appeared at
a similar temperature (46.9ºC) than the corresponding steps for the functionalized
membranes (Table 1) due to the low content of C-Cl dipoles present in the M-Cl
membrane.
The thermal stability of the membranes was evaluated using TGA and Table 1 shows
the thermal data, in terms of characteristic weight loss (T5 and T10 respectively).
1.4
175 o
C EI-MS m/z: 36 (Cl)
M-Cl Tg= 46.9oC
M-5b Tg= 45.4oC
M-Cl
M-5b
100
80
60
40
20
0
0.0025
0.0020
0.0015
0.0010
0.0005
B
C
A
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.12
-0.13
-0.14
-0.15
425 o
C
M-Cl
M-5b
38
40
42
44
46
48
50
100 200 300 400 500 600 700 800
100 200 300 400 500 600 700 800
Temperature (oC)
Temperature (oC)
Temperature (oC)
Fig.3. DSC thermograms (A), TGA and DTGA curves (B) and evolved gas analysis by
MS detector (C) of the M-Cl crosslinked membrane and M-5b membrane functionalized
with naphthalimide derivative 5b.
Figure 3B shows the TGA thermograms obtained for the M-Cl and M-5b membranes
and their corresponding first derivative curves. The M-Cl membrane exhibits a weight
loss of 2.8% that was identified by MS analysis of evolved gas at 175ºC as chlorine (EI-
MS m/z: 36 (Cl)), as it is shown in fig 3C. After functionalization, the chlorine weight
was not observed confirming the total hydrolysis of unreacted acid chloride. For all
functionalized membranes two peaks were observed, as it is shown for M-5b in Fig. 3B.
The highest weight loss rate that was observed at 410 °C corresponds to the degradation
of acrylic components of the membrane and the second one observed at 431 °C has been
attributed [7] to the lactam subgroup degradation.
The swelling properties of the membranes were examined by measuring the swelling
(%) after immersion in water and THF and are detailed in Table 2.
18

ACCEPTED MANUSCRIPT
Table 2. Swelling degree (SD) in water and THF, advancing contact angle (CA) and
total (γ), polar (γp), and disperse (γd) surface energy data for the crosslinked membranes.
SD (%)a
Static CA (°)
Surface Energy (mN.m-1)b
Membrane
Water
THF
45.4
61.2
62.0
63.5
Water
Diiodomethane
γ
γp
γd
M-Cl
M-4a
M-5b
M-6b
29.8
29.9
30.4
29.9
59.9
69.6
71.7
71
41.6
42
38.6
46.1
51.64
45.43
45.75
46.22
13.48
9.25
6.56
6.88
38.16
36.18
39.19
39.34
a±0.3; b Calculated using Owens-Wendt´s method
The SD in water (approx. 30 %) and in THF (approx. 60 %) confirms the hydrophilic
character of the membranes and the adequate swelling for solid sensors in water and
organic media. Also, the hydrophilicity of the membrane surfaces was characterized by
static contact angle determination. The measured CAs were then used to calculate the
polar, disperse, and total surface energies of the membranes. The change in the total
surface energy (γ) and its polar (γp) and dispersive (γd) components of the M-Cl and
functionalized membranes with naphthalimides (M-4a, M-5b and M-6b) was calculated
by the Owens–Wendt's method [28] (Table 2). The variation in the values of the total,
polar, and disperse solid surface energies before (M-Cl) and after functionalization (M-
4a, M-5b and M-6b), shows that the total surface energy of the functionalized
membranes decreases from 51.64 mN·m−1 for M-Cl to 45-46 mN·m−1 for the
membranes containing naphthalimide moieties and, particularly for the polar
component.
The functionalized membranes with piperazine-naphthalimide groups were easily
handled materials and exhibited convenient properties for solid sensor applications,
such as swelling in water, thermal stability, flexibility, optical transparency and
dimensional stability to be reused. The obtained three-dimensional cross-linked
hydrophilic polymer networks are capable of swelling or de-swelling reversibly in water
and retaining a controlled and reproducible volume of liquid in the swollen state. This
behaviour, as will be seen in the next section, allowed controllable responses to external
environmental changes of pH.
3.3. Effect of pH on the absorption and fluorescence properties of piperazine
naphthalimides derivatives and membranes
19

ACCEPTED MANUSCRIPT
Piperazine substituted naphthalimides such as the structures synthetized in this work
(Fig. 2) exhibit interesting fluorescence properties, hence 4-amino-1,8-naphthalimide
fluorophore can be quenched by the PET process that occurs from the alkylated amine
donor to the 4-amino-1,8-naphthalimide fluorophore through the piperazinyl ring. The
fluorescence of the 4-amino-1,8-naphthalimide fluorophore is quenched. The PET path
can be switched off either by the protonation or quaternization of the amine and the
fluorescence of the naphthalimide fluorophore is then recovered as is shown for the
derivative 4a anchored to the membranes in figure 4.
Fig.4. Fluorescence response of piperazine-naphthalimide structure to pH though PET
mechanism.
The effect of pH variation on the absorbance and fluorescence properties of these
derivatives is illustrated in figure 5 for the naphthalimide derivative 4a in solution and
the corresponding membrane M-4a under the same conditions.
20

ACCEPTED MANUSCRIPT
2.0
3.6
4.3
5.2
6.0
6.5
6.8
7.2
7.6
8.0
8.5
9.0
-
2.0
-
M-4a
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.6
0.4
0.2
0.0
4a
[OH ]
[OH]
3.6
4.7
5.6
6.0
6.3
6.7
6.8
7.0
7.5
8.2
8.8
9.4
10.8
12.0
9.5
10.0
10.5
11.0
11.5
12.0
350
400
450
500
550
350
400
450
500
550
Wavelength(nm)
Wavelength(nm)
600
500
400
300
200
100
0
2.0
3.0
4.0
5.0
5.6
6.3
6.8
7.3
8.0
8.7
8.9
9.5
10.0
11.0
12.0
2.0
3.6
4.7
5.4
6.0
6.3
6.6
6.9
7.1
7.5
8.1
8.7
9.4
10.7
12.0
4a
M-4a
-
-
300
200
100
0
[OH ]
[OH ]
500
600
700
500
600
700
Wavelength(nm)
Wavelength(nm)
Fig. 5. Spectroscopic characteristics as a function of pH (2.0-12.0) of absorption and
fluorescence of naphthalimide derivative 4a and M-4a membrane.
All the absorption spectra of the naphthalimide derivatives and membranes exhibit (as
solution or as 180µm-membrane) similar band shapes in the absorption range of 350-
500 nm. As it is shown in figure 5, for the compound 4a and membrane M-4a, their
absorption peaks were red-shifted from 388 to 408 nm with a clear isosbestic point at
402 nm when pH values were changed from 2.0 to 12.0 in water:ethanol (4:1) by
adjusting pH values with NaOH and HCl aqueous solutions. As expected, the
fluorescence intensities originating from 4a and M-4a fluorophores were varied upon
different pH values (Figure 5), resulting in a strong enhancement in acid media due to
the protonation of the piperazine and its subsequent PET blocking effect (Fig. 4). In the
case of compound 4a the factor of enhancement (FE) was about 25 times in
fluorescence intensity when adjusting the pH from 12.0 to 2.0 (Table 3). Moreover, the
21

ACCEPTED MANUSCRIPT
wavelength emission maxima were red-shifted by around 20 nm (Table 3) by changing
the pH values from 2.0 to 12.0 for all the naphthalimide derivatives in solution except
for naphthalimide 3b and 4b that do not have hydroxyl substituents in their structures
and the red-shift for them was about 2-3 nm. For the naphthalimide fluorophores
bonded to the membranes though the hydroxyl groups, the bathochromic shift of the
fluorescence maxima with pH was also 2-3 nm.
All the spectral data measured as a function of pH values for the naphthalimide
derivatives and the three functionalized membranes are summarized in Table 3.
Table 3. Various parameters for piperazine-1,8-naphthalimide derivatives (a and b
structures) in solution(a) and bonded to the polymer membrane (M) determined
according to pH dependence by UV–Vis absorption and fluorescence spectroscopy.
Parameter
3a
4a
5a
6a
3b
4b
5b
6b
M-4a
M-5b
M-6b
389
4.07
403
413
4.04
8.24
533
388
4.06
402
408
4.02
7.37
529
388
4.05
402
388
4.06
402
389
4.06
404
389
4.06
402
389
4.056
402
388
4.06
402
388
-
403
407
-
7.62
502
-
388
-
402
408
-
7.25
503
-
390
-
402
408
-
6.40
503
-
λABS-acid
Log εacid
λIsobestic
(b)
408
408
410
409
409
409
λABS-base
Log εbase
(b)
4.01
6.99
530
0.184
0.101
4.02
6.65
530
0.238
0.099
4.01
8.62
531
0.084
0.079
4.02
7.30
529
0.274
0.249
4.02
6.82
530
0.170
0.095
4.02
6.71
529
0.197
0.091
(c )
pKa
λFLU-acid
(d)
0.097 0.330
0.093 0.304
φFLU-acid
φFLU
(d)
-
-
-
pH=7
(d)
(d)
0.017 0.025
0.203 0.389
543
0.010 0.013
7.1
0.022
0.285
550
0.010
6.95
0.026
0.293
550
0.009
6.4
0.026
0.213
532
0.005
7.15
0.020
0.365
532
0.010
7.11
0.017
0.270
554
0.009
7.04
0.024
0.275
545
0.007
6.48
water:ethanol (4:1, v/v)<
< Ethanol <<< hexane
505
-
φFLU ethanol
φFLU hexane
λFLU-base
548
506
-
6.71
504
-
6.5
( d)
φFLU-base
*
(c )
6.93
7.15
pKa
(e )
6329
8.5
6258
25.3
6070
19.1
6061
22.0
6293
18.1
6123
17.9
6070
22.0
5998
27.1
5515
12.3
5618
8.0
5539
9.5
∆ν
FE (f )
(a) Measured at 10-4 M in water-ethanol (4:1, v/v) unless specifically stated otherwise. The subscripts “acid” and
“base” refer to the limiting value of a given parameter when the acid or base condition is increased until pH=2.0 or
pH=12.0 respectively. Fluorescence emission spectra were obtained by excitation at λIsobestic ; (b) ε, molar absorptivity
in M-1cm-1; (c) Obtained [31] by analyzing the pH dependence of the absorbance (A) or emission intensity (I) at a given
wavelength according to the equation log[(Aacid − A)/(A − Abase)] = pH − pKa or log[(IFLU-acid − I)/(I − IFLU-base)] = pH -
*
pKa
(d) Relatively to coumarin 6 (
ϕF=0.78 in ethanol), uncertainty = 0.001 or 10%, whichever is the larger; (e)
Stokes shift (νFLU - ν
ABS) in frequency (cm-1); (f) Acid- induced fluorescence enhancement factor FE= AFLU-acid/AFLU-.base
The quantum yields of fluorescence in acid media (Table 3) for the hydroxy alkyl
substituted piperazine naphthalimides derivatives (φFLU-acid 0.33-0.084) are lower than
=
that of the non-hydroxylated derivative N-butyl-4-N′-methylpiperazino-1,8-
22

ACCEPTED MANUSCRIPT
naphthalimide (φFLU-acid
=0.55) [22]. Also, the quantum yields of fluorescence in basic
media (Table 3) are higher (φFLU-base = 0.005-0.010) than that measured for N-butyl-4-
N′-methylpiperazino-1,8-naphthalimide (φFLU-base = 0.001) [22]. The quantum yield of
fluorescence for the membranes couldn’t be calculated due to the difference in geometry
between the fluorescence standard in solution and the membranes. The factor of
fluorescence enhancement for the membranes in acid media was calculated (Table 3) by
comparing the increase of intensities from basic to acid media and the enhancement was
around 8-12 times. This enhancement in acid media (FE = 8-12.3) is higher than that
obtained by other authors [23], where an enhancement of 4.7 was reported with a
membrane obtained by copolymerization of N-allyl-4-(4´-methyl-piperazinyl)-1,8-
naphthalimide with 2-hydroxyethyl methacrylate and acrylamide.
The presence of hydroxyl groups in the naphthalimide derivatives studied here and the
ester functionality as a linking group of the naphthalimides in the membranes reduced
the factor of enhancement of fluorescence in acid media with respect to that for other
similar structures having only N-alkyl substituents [21,22]. Even though FE values
obtained with the membranes confirm their interesting proton “off–on” switch
behaviour as an optical pH solid sensor.
The pKa values of naphthalimide derivatives and the functionalized membranes were
calculated [31] from the curves of absorption changes in the presence of increasing
acidity (from pH= 12.0 to 2.0) and the obtained values are compiled in Table 3.
The smaller substituents on the amine groups (structures 3 with hydrogen or 4 with
methyl as substituents) enabled a higher pKa as they are more easily protonated than the
bigger substituents (structures 5 and 6). The pKa values obtained for the functionalized
membranes are close to those values measured for the corresponding piperazine
derivatives (Table 3) confirming a similar sensitivity to acid media. The pKa of the
functionalized membranes is close to the pH of 7.0 which makes it adequate for sensors
in biological and environmental applications [19].
The fluorescence response of the piperazine derivatives and the functionalized
membranes at different pH was compared and the results are plotted in figure 6.
23

ACCEPTED MANUSCRIPT
14
M-4a
M-5b
M-6b
3a
350
300
250
200
150
100
50
4a
12
5a
6a
10
8
3b
4b
5b
6b
6
4
2
0
0
2
4
6
8
10
12
2
4
6
8
10
12
pH
pH
Fig.6. Comparison of the fluorescence intensity changes (I/Io) with pH (2.0-12.0) for the
piperazine-naphthalimide derivatives in solution (A) and for functionalized membranes
(B). (I0 and I are the fluorescence intensities of initial (pH=12.0) and that after
acidification respectively).
All the synthesized naphthalimides (Fig. 6A) exhibited a sensitive fluorescence change
towards pH but with different intervals and slopes of changes depending of the size of
the amino substituents. For the functionalized membranes (Fig. 6B) the fluorescence
intensity increased as the pH values decreased from 9.0 to 5.0 for M-4a and from 8.5 to
4.0 for the membranes obtained by linking the naphthalimides to the substituent of the
piperazine structure, M-5b and M-6b. In the acidic pH range, especially for pH lower
than 4.0, the fluorescence intensity attained its maximum and kept unchanged as well as
in the basic pH range up to 9.0 where the fluorescence intensity reached its minimum.
The pKa* values (Table 3) for the excited state do not differ strongly from the
corresponding pKa for the ground state except for the structures 3a and 3b where the
substituent of the piperazine is a hydrogen. For the rest of the structures and membranes
the position of the inflection points in the fluorometric titration is similar to those
obtained from the absorbance changes with pH.
The fluorescence quantum yields of the naphthalimides derivatives studied here are
relatively low in polar solvents (water, ethanol) in comparison to those values obtained
in non-polar solvents such us hexane (Table 3). This fact is probably due to the photo-
induced electron transfer from the piperazine to the fluorophore and the fact that in
polar solvents the orbit energy of HOMO becomes lower facilitating the PET process.
24

ACCEPTED MANUSCRIPT
The polarity effect on the photophysical properties of the naphthalimide derivatives was
studied by measuring their absorption and fluorescence spectra in solvents of increasing
solvent polarity parameter ET(30). All the naphthalimide derivatives exhibited similar
solvathochromic UV-Vis and emission spectra. As an illustrative example in figure 7,
the values of absorption and emission maxima are plotted for different solvents.
550
500
450
400
absorbance
fluorescence
550
500
450
400
absorbance
fluorescence
M-5b
5b
11
12
13
10
7
11
8
9
10
13
12
9
5
7
8
6
4
2
56
4
1
3
23
1
30
35
40
45
50
55
30
35
40
45
50
55
ET (30)
ET (30)
Fig. 7. Dependence of absorbance and Fluorescence emission wavelengths maxima of
naphthalimide derivative 5b and the membrane M-5b on the empirical solvent polarity
parameter, ET(30). Solvents: 1. Hexane, 2. Toluene, 3. Diethyl ether, 4. Dioxane, 5.
THF, 6. Ethyl acetate, 7. Chloroform, 8. Acetone, 9. Acetonitrile, 10. 2-propanol, 11.
Butanol, 12. Ethanol, 13. Methanol.
The maximum wavelengths of the absorption and fluorescence emissions are slightly
red shifted with increasing solvent polarity except for the H-bonding solvents in the
case of naphthalimide 5b. This is due to the presence of hydroxyl groups in the structure
that provoke specific solute-solvent interaction. In general, the absorption maxima are
less sensitive to the influence of solvent polarity than the fluorescence maximum. In the
membranes, as shown in Fig. 6 for M-5b, the low bathochromic shifts in the
fluorescence maximum with an increase of solvent polarity are consistent with small
differences between the dipole moments of the ground and excited states.
25

ACCEPTED MANUSCRIPT
3.4. Response time to pH of the functionalized membranes
The reversibility of the pH induced fluorescence signal was evaluated by determining
the absorbance and fluorescence of the membranes by alterning their immersion in
solutions of pH 2.0 and pH 12.0. Several cycles of successfull enhancement/quenching
of fluorescence were carried out with each membrane without loss of fluorescent
emission. The protonation of the aliphatic amine group involved seems quite reversible
and no noticeable hysteresis effect was observed in the solid sensors.
The pH response of the membranes is dependent upon the proton permeability into the
photocrosslinked material. Hence, the response time of the absorption and fluorescence
spectra of the functionalized membranes was studied and the obtained results are plotted
in Fig. 8.
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
A
C
B
M-4a
M-5b
M-6b
M-6b 180
M-6b 90
µm
M-4a
M-5b
M-6b
µm
ADIC MEDIA
ACID MEDIA
BASIC MEDIA
0
10
20
30
40
0
10
20
30
40
0
10
20
30
40
Time (min)
Time (min)
Time (min)
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
D
F
E
M-4a
M-5b
M-6b
M-4a
M-5b
M-6b
M-6b 180
M-6b 90
µm
µm
BASIC MEDIA
ADIC MEDIA
ACID MEDIA
0
10
20
30
40
0
10
20
30
40
0
10
20
30
40
Time (min)
Time (min)
Time (min)
Fig. 8. Time response to pH of the functionalized membranes: Absorbance (A) and
fluorescence (D) to acid media (pH=2.0), absorbance (B) and fluorescence (E) to basic
media (pH=12.0) and effect of the membrane thickness in time response to acid media
in absorbance (C) and fluorescence (F).
26

ACCEPTED MANUSCRIPT
The pH response of the membranes is dependent upon the proton permeability into the
crosslinked matrix and the three membranes exhibited a high sensitivity to pH in water.
The absorbance and fluorescence of the membranes to acidic media (Fig. 8A and 8D
respectively) reached the equilibrium in less than 15 min. The sensitivity to the basic
medium of the membranes (Fig. 8B and 8E) is lower than that observed in acid media
showing a slow response of around 20 min. Hence, the protonation process of the
aliphatic piperazine group involved seems to be slower than the deprotonation process.
The time response of the membrane M-4a is shorter that those of the membrane b type.
In the membrane M-4a the naphthalimide sensing functionality is linked to the polymer
matrix through the imide group and the piperazine group is far from the polymer main
chain. Hence, the interaction of the amino group of the piperazine with a proton is much
easier than those of M-5b and M-6b where the piperazine is closer to the polymer main
chain that can impair the mobility and hence the interactions of the sensing moiety.
The sensitivity of the solid sensor is directly related to the thickness of the pH sensitive
membrane. To see the influence of the membrane thickness in the time response to the
pH of the sensor, we prepared a membrane under the same experimental conditions but
with 90 microns of thickness that was also an easily handled material and with good
physical properties. As expected, the time response of this M-6b 90 µm-membrane
(Fig. 8C and 8F) was notably shorter than that of the corresponding M-6b 180 µm-
membrane.
In this study, we have described a useful procedure to obtain a water-swollen
photocrosslinked membrane containing acid chloride groups capable of reacting with
sensing molecules containing adequate functionalities, such as hydroxyl groups. The
functionalization of M-Cl membrane was carried out using three derivatives of
piperazine naphthalimide selected from a series of compounds that had been previously
synthetized and spectrophotometrically characterized as a function of pH variations.
The dependence of the pH sensitivity was related to the size and structure of the
piperazine-naphthalimide substituents linked to the membrane. The solid sensors
functionalized with naphthalimides acted as an acid sensor with interesting ranges of
sensitivity to pH changes, from 9.0 to 4.0, which are adequate for medical and
environmental sensing applications.
27

ACCEPTED MANUSCRIPT
4. Conclusions
In summary, we have developed a procedure to obtain, using a photopolymerization
reaction, water-swollen membranes capable of functionalization with naphthalimide
fluorescence sensors and with a pH response.
The obtained results in the synthesis of the series of substituted piperazine
naphthalimide derivatives carried out in this work suggested that microwave-assisted
syntheses led to higher yields within very short reaction times in comparison to the
conventional methods.
The new solid sensors exhibited controlled swelling in water (30%) and sensitive
fluorescence changes towards pH between 9.0 and 4.0, and showed a very strong
fluorescence at pH < 5.0.
The time response of the functionalized membranes to pH is dependent on the thickness
of the membrane, the size of the substituents and the naphthalimide position of linkage
to the membrane. The pH induced fluorescence signal of the solid sensors is reversible
and several cycles of enhancement/quenching of fluorescence were successfully carried
out without a noticeable hysteresis effect.
The present functionalized membranes could be applied as “Off-On” pH-fluorescence
sensor in medical and environmental applications with satisfactory results.
Acknowledgments
This study was supported by MINECO (Project Ref. MAT2012-31709). S. Fernández-
Alonso thanks MINECO for a pre-doctoral fellowship linked to the project.
References
[1] C. McDonagh, C.S. Burke, B.D. MacCraith, Optical chemical sensors, Chem. Rev. 108 (2008) 400–
422.
[2] H.H. Qazi, A.B.B. Mohammad, M. Akram, Recent progress in optical chemical sensors, Sensors 12
(2012) 16522–16556.
[3] S.M. Borisov, O.S. Wolfbeis, Optical biosensors, Chem. Rev. 108 (2008) 423–461.
28