Fluorometric sensing of hydroxylamine in an aqueous medium utilizing a diphenyl imidazole-based probe

Article abstract of DOI:10.1039/d0ob00608d

The detection of hydroxylamine in an aqueous medium is challenging due to its very similar chemical reactivity to its nearest competitors such as hydrazine hydrate and primary amines. Moreover, the detection of hydroxylamine at neutral pH adds further com

Full text of DOI:10.1039/d0ob00608d

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DOI: 10.1039/D0OB00608D
Fluorometric sensing of hydroxyl amine in aqueous medium utilizing a diphenyl imidazole-
based probe
Punam Rana,a Lipsarani Panda, a Narayan Murmu,a Bhawani Prasad Bagb and
Satya Narayan Sahu*a
a School of Chemistry, Sambalpur University, Jyoti Vihar, Burla-768 019, Odisha, India
Email: snsahu.chem@gmail.com; snsahu@suniv.ac.in
b Department of Biotechnology and Bioinformatics, Sambalpur University, Jyoti Vihar, Burla-768 019,
Odisha, India
Graphical Abstract
H
N
N
NH2OH
pH =7.4
95% H2O
C
CHO
N
H
N
H
N OH
Non fluorescent
Highly fluorescent
Abstract
The detection of hydroxyl amine in aqueous medium is challenging due to its very similar
chemical reactivity with nearer competitors such as hydrazine hydrate and primary amines.
Moreover, the detection of hydroxyl amine at neutral pH adds further complexity to the sensing
phenomenon due to its poor reactivity in neutral aqueous medium. In this work, we have
presented a diphenyl imidazole benzaldehyde (DIB) probe which demonstrates the detection of
hydroxylamine (HA) in micromolar concentration with high selectivity in 5% DMSO phosphate
buffer solution at pH 7.4 via a fluorescence “turn-on” signal. The interaction of hydroxyl amine
with the probe has been comprehensively studied by using fluorescence technique, proton NMR,
FTIR, ESI-Mass and DLS measurements. The experimental results were further corroborated
with the DFT studies. These results could pave the way toward the development of molecular
indicators for hydroxyl amine in chemical and biological platforms.
1. Introduction
Hydroxylamine (HA), NH2OH, is an oxidised form of ammonia which is abundantly used by
various industries for processing of dyes, textile, plastics, semiconductors and photography.1,2
Further, it is one of the important chemical constituent for the synthesis of caprolactam, a
1

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monomer for the production of Nylon-6 based products.3 Very recently, it is used as a primary
material for making of hydroxylamine nitrate (HAN) which is considered as the next generation
nontoxic propellant as an alternative to toxic hydrazine based propellants to be employed in
spacecraft, nuclear power and military services.4 Besides, it serves as an active ingredient for the
synthesis of hydroxamic acid/hydroxamate based drug substances.5 Moreover, HA can act as
nitric oxide (NO) donor molecule and involve in many biological functions.6,7 Despite its
widespread applications, unfortunately HA is a potentially hazardous material which can elicit
highly specific mutations with cytosine8 and thus can cause modest level of toxicity to humans,
animals and plants. 2 μg/day is the maximum recommended exposure limit for hydroxylamine.9
Consequently, many research groups have taken an utmost interest to develop a selective and
sensitive analytical procedure for the trace level estimation of hydroxylamine in aqueous
medium. Although a diverse range of conventional techniques such as high-performance liquid
chromatography, gas chromatography, potentiometric, polarographic, biamperometric and
electrochemical methods have been developed,10-17 but these practices do not offer an
economical and practical monitoring of HA at the desired location. In this context, fluorescence
technique could provide a rather precise substitute owing to its several benefits such as fast,
sensitive, selective and operational simplicity for detection and quantification of hydroxylamine.
In addition, the detection of hydroxyl amine is quite challenging due to very similar chemical
reactivity with its close competitors such as hydrazine hydrate and primary amines.18-20 Although
many reports have been published on the detection of hydrazine,21,22 however, to our best only
two reports have been published by Sedgwick et. al. and Dong et. al. for selective fluorogenic
detection of hydroxyl amine.23,24 Therefore, development of molecular fluorophores for specific
and sensitive detection of HA in micromolar concentrations has received a great deal of interest.
With this vision and in continuation to our efforts toward the development of chromofluoroscent
molecular sensors,25-27 we have prepared
a
probe 4-(4,5-diphenyl-1H-imidazol-2-
yl)benzaldehyde (DIB) which demonstrates the detection of hydroxylamine (HA) in micromolar
concentration with high selectivity in 5% DMSO phosphate buffer solution at pH 7.4 via a
fluorescence “turn-on” behavior. The probe elicits a visual colour change from colourless to cyan
fluorescence to the sensor solution in presence of HA. Further, the probe shows a prominent
fluorescence “turn-on” signal with HA in comparison to its closer competitor hydrazine.
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CHO
CHO
CHO
N
O
O
CH3COONH4
CH3COOH
+
NH
Reflux
120oC
DIB
Scheme 1. Synthesis protocol for probe DIB
2. Results and Discussion
2.1. Synthesis and characterization of probe DIB
The general experimental methods, preparation of test samples and synthetic procedure of probe
4-(4,5-diphenyl-1H-imidazol-2-yl)benzaldehyde (DIB) (Scheme 1) has been described in
Supporting Information (see ESI). Condensation reaction of benzil with terephthaldehyde in
glacial acetic acid medium affords DIB in moderate yield. The analytical characterization of DIB
was consistent with its indicated structure (Fig. S1-S4). The proton NMR spectrum (Fig. S1) of
DIB showed a singlet at 13.03 ppm which could be assigned to imidazolyl-NH proton that was
exchanged upon addition of D2O. The singlet at 10.03 ppm could be attributed to aldehydic –
CHO proton. The doublets at 8.30 and 8.01 ppm and multiplets at 7.58-7.23 ppm conform to the
aromatic protons. The 13C NMR shows the characteristic signal at 193 ppm for the carbonyl
carbon of the aldehyde group (Fig. S2). The FTIR spectra (Fig. S3) shows bands at 3355, 1695
and 1442 cm-1 which respectively corresponds to the secondary amine –NH, >C=O and Ar-C=C-
stretching frequencies. The ESI-MS mass data (Fig. S4) further confirmed the formation of probe
DIB by the appearance of a signal at m/z 325.33 for the [M+H]+ ion.
2.2. Photophysical studies of DIB
The probe DIB comprises an imidazole ring conjugated with a benzaldehyde unit. Thus it is
expected that the imidazole unit and the aldehyde group behave as a donor and acceptor
combination respectively which could show significant photophysical properties. We first
studied the solvatofluorism of the probe in eight different organic medium such as benzene,
toluene, tetrahydrofuran, 1,4-dioxane, acetone, methanol, acetonitrile and dimethylsulphoxide
(DMSO) of varying polarity at 50 µM concentration at room temperature. The solutions of DIB
in organic solvents exhibited a strong photoluminescence under UV light at 365 nm (Fig. S5).
The fluorescence spectra of the probe exhibited a gradual bathochromic shift in its emission band
by increasing the solvent polarity (Fig. 1a). In non-polar toluene, the probe exhibited an emission
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band at 425 nm while in polar methanol medium it appeared at 575 nm with a significant
bathochromic shift (Δλem) of 150 nm. This solvent depended fluorescence behaviour clearly
suggests that the photoexcited states of DIB are polar in nature which gets stabilized by solvation
resulting in light emission at a higher wavelength from the stabilized molecules.28,29
1.2
Benzene
Toluene
THF
(a)
1.0
Dioxane
Acetone
0.8
Methanol
Acetonitrile
DMSO
0.6
0.4
0.2
0.0
400
450
500
550
600
650
700
Wavelength (nm)
1.0 mM
(b)
0.5 mM
0.1 mM
400
300
200
100
0
400
450
500
550
600
650
Wavelength (nm)
(c)
% H2O
2000
1500
1000
500
0
0
10
20
30
40
50
60
70
80
90
100
400
450
500
550
600
650
Wavelength (nm)
Fig. 1. (a) Normalized emission spectra of DIB (50 µM) in benzene, toluene, THF, dioxane, acetone,
methanol, acetonitrile and DMSO. (b) Fluorescence spectra of DIB at various concentrations in DMSO.
(c) Fluorescence spectra of DIB (50 µM) at various DMSO-Water fractions (λex = 365)
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The shifts in emission bands could be assigned to the π-electron delocalization from the
imidazole donor to aldehyde acceptor via a “push−pull” mechanism within the DIB molecule as
a result of increasing solvent polarity. Further, aggregation induced emission (AIE) and
aggregation caused quenching (ACQ) characteristics of DIB have been evaluated by
fluorescence studies. For the ACQ study, fluorescence spectra were measured at various
concentrations of DIB ranging from 1.0 mM to 0.1 mM. It was observed that, with increase in
probe concentration the fluorescence intensity decreases which indicates that the concentrated
probe solution is poorly emissive in nature (Fig. 1b). In contrast, the AIE behavior of DIB was
examined by recording the emission spectra at varying DMSO-water fractions (fw = 0 to 100%)
at 50 µM probe concentration. It was observed that the cyan colour fluorescence signal goes on
decreasing with increase in water fraction (fw) when analysed under an UV lamp at 365 nm (Fig.
S6). The fluorescence spectra of the corresponding solutions showed a regular decrease in the
fluorescence intensity at 490 nm with increase in water fraction (fw) from 0 to 100% (Fig. 1c).
This result clearly suggested the absence of AIE behavior in the probe.30
2.3. Fluorescence analysis of DIB in presence of various amines
The interaction of DIB (50 µM) with various amines such as urea, thiourea, aniline,
triethylamine (TEA), ammonium hydroxide, hydroxyl amine, ethylene diamine, piperidine,
hydrazine hydrate and methyl amine dissolved in Millipore water, was analysed in 5% DMSO
phosphate buffer solution at pH 7.4. By addition of 20 equiv. of different amines to the probe
solution, intense cyan colour fluorescence was appeared only in presence of hydroxyl amine
(HA) (Fig. 2a). On the other hand, addition of hydrazine hydrate (HH) resulted in a faint blue
fluorescence coloration while other amines showed insignificant fluorescence colour change in
the probe solution when observed under UV-light at 365 nm (Fig. 2a). The fluorescence spectra
of the corresponding solutions showed more than 30-fold enhancement in emission intensity at
465 nm in presence of HA while HH enhances the intensity to 8-fold only (Fig. 2c). In contrast,
other amines did not exhibit any significant enhancement in fluorescence intensity (Fig. 2b,c).
These results clearly indicate a greater selectivity of DIB toward HA in comparison to its closer
competitor hydrazine hydrate (HH) via a change in fluorescence signal. Further, the fluorogenic
selectivity of DIB against various anions and cations were investigated by visual observation
under an UV-light at 365 nm. Upon addition of 20.0 equiv. of different anions and cations such
as F¯, Cl¯, Br¯, I¯, H2PO4¯, HSO4¯, OH¯, AcO¯, CN¯, S2-, Ag+, Ba2+, Ca2+, Cd2+, Co2+, Cu2+,
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Cr2+, Fe2+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+, Pd2+ and Zn2+, no significant fluoDreOsI:c10e.n10c3e9/Dc0oOlBo00u6r08D
change was detected in the probe solution (Fig. S7).
(i)
(ii) (iii) (iv) (v) (vi) (vii) (viii) (ix) (x) (xi)
(a)
DIB+HA
(b)
(c)
30
20
200
150
100
50
DIB+HH
10
0
0
r
400
450
500
550
600
650
a
e
a
n
p
h
h
d
h
B
I
T
U
P
T
E
H
A
A
M
H
D
Wavelength (nm)
+
+
+
+
+
+
+
+
+
+
B
I
B
I
B
I
B
I
B
I
B
I
B
I
B
I
B
I
B
I
D
D
D
D
D
D
D
D
D
D
DIB with various species
Fig. 2. (a) Visual fluorescence change of DIB (50 µM) in 5% DMSO phosphate buffer solution at pH
7.4 with various amines (20 equiv.) under UV-light at 365 nm. (i) Probe DIB alone and probe with other
species (ii) aniline (An) (iii) methyl amine (Ma) (iv) piperidine (Pp) (v) hydroxyl amine (Ha) (vi) triethyl
amine (Te) (vii) hydrazine hydrate (Hh) (viii) thiourea (Th) (ix) urea (Ur) (x) ethylene diamine (Ed) (xi)
ammonium hydroxide (Ah). (b) Fluorescence spectra of DIB (50 µM) in 5% DMSO phosphate buffer
solution at pH 7.4 in presence of 20 equiv. of various amines. (c) Emission intensities (I/I0) at 465 nm of
corresponding spectra in presence of various species. (λex= 365 nm).
2.4. Effect of pH on the fluorescence properties of DIB in presence of hydroxyl amine
It is well known that the interaction of aldehyde group with hydroxyl amine is highly pH
depended.31 Thus, to analyze the effect of pH on the fluorescence properties of DIB (50 µM) in
presence of HA (20 equiv.), the fluorescence spectra were recorded over a pH range of 1 to 13. It
was observed that, probe DIB showed no significant change in its emission intensity at 465 nm
over the pH range of 1 to 13 which indicates its good stability over the tested pH (Fig. S8).
However, addition of HA enhances the fluorescence intensity of the probe at 465 nm and
exhibited a stable response over a pH range of 4-9 (Fig. S8). At pH 2 and 3, the emission signal
of the probe at 465 nm is quite high in presence of HA which could be due the favorable reaction
condition between the aldehyde and NH2 group at acidic conditions.31 In contrast, fluorescence
intensity of the probe at 465 nm decreases beyond pH 9. These findings suggested that the probe
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can detect HA over a wide range of pH of 4 to 9. Although the probe showDeOdI: 1a0.1s0i3g9/nDi0fOicBa00n6t08D
fluorescence response toward HA at pH 2-3, we have not taken this pH range to study the
recognition experiments due to highly acidic condition. Rather, the physiological pH medium at
7.4 was considered throughout all the recognition experiments. Moreover, a 5% DMSO
phosphate buffer solution at pH 7.4 was used for all the studies.
2.5. Optical response time of DIB with hydroxyl amine and hydrazine hydrate
In order to establish the optical response time, the time-dependent single wavelength
fluorescence measurement was performed for DIB in presence of HA and HH at 465 and 458 nm
respectively. The emission intensity of DIB with hydroxyl amine (at 465 nm) and hydrazine
hydrate (at 458 nm) increases with time and finally reached a plateau beyond 2 hour (Fig. S9).
However, the emission enhancement in presence of HA was found to be much higher than with
HH even at the earlier period of time. By analyzing the fluorescence intensities at the initial 30
minutes period, it was observed that the intensity for HA is much higher and fast responsive in
comparison to HH (Fig. S9). This observation clearly demonstrates that probe DIB shows a
faster response and higher sensitivity toward HA in comparison to its closer competitor HH.
2.6. Fluorescence titration study of DIB with hydroxyl amine and hydrazine hydrate
From the fluorescence time response study (Fig. S9), a better sensitivity was established for HA
in comparison to HH by the probe DIB. Thus, to get insight into the sensing mechanism,
fluorescence titration spectra were recorded by the addition of different equivalents of HA and
HH to DIB (50 µM) in 5% DMSO phosphate buffer solution at pH 7.4 with incubation time of 2
hour. Addition of HA to DIB increases the fluorescence intensity at 465 nm concomitantly with
various equivalents up to more than 30-fold enhancement (inset Fig. 3a). Further, UV-
illumination at 365 nm to different vials containing DIB (50 µM) with various equivalents of HA
shows a gradual increase in cyan colour fluorescence with increasing equivalents of HA that can
be easily distinguish by visual observation (Fig. S10). On the other hand, a similar set of titration
experiment of DIB with HH showed the increase in fluorescence intensity at 458 nm with
continual addition HH exhibiting an enhancement of 8-fold only even after the addition of 20
equivalents (Fig. 3b). The fluorescence enhancement of DIB in presence of HA is significantly
higher in comparison to HH which indicates a higher recognition affinity of the probe toward
hydroxyl amine. Moreover, the concentration-dependent fluorescence response for both HA and
HH shows a linear relationship up to 20 equivalents (Inset of Fig. 3a,b). The binding constant
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(Ka) of probe DIB toward HA and HH were calculated from their fluorescence tiDtrOaI:ti1o0.n103p9r/Do0fOilBe00a6t08D
465 and 458 nm respectively by using the linear regression analysis. The binding constant (Ka)
for HA and HH were found out to be 2.23 x 104 M-1 and 3.12 x 103 M-1 with regression
coefficient of 0.98 and 0.99 respectively (inset Fig. 3a,b). The higher binding constant of DIB for
HA in comparison to HH clearly signifies a high level of sensitivity of the probe toward HA.
Finally, the detection limit of DIB (50 µM) for HA was determined by fluorescence
measurement at 465 nm in 5% DMSO phosphate buffer solution at pH 7.4 and was found out to
be 28 µM (̴ 0.92 ppm) (Fig. S11).
R2 = 0.99
(b)
R2 = 0.98
Ka = 2.23×104 M-1
(a)
Ka = 3.12×103 M-1
6
4
2
0
30
20
10
0
60
40
20
0
200
150
100
50
0
5
10
15
20
0
5
10
15
20
Equivalent of HA
Equivalent of HH
0
400
450
500
550
600
650
400
450
500
550
600
650
Wavelength (nm)
Wavelength (nm)
Fig. 3. (a) Fluorescence titration spectra of DIB (50 µM) in 5% DMSO phosphate buffer solution at
pH 7.4 with of 0.0 to 20.0 equivalents of HA. Inset shows the fluorescence intensity at 465 nm vs. equiv.
of HA. (b) Fluorescence titration spectra of DIB (50 µM) in 5% DMSO phosphate buffer solution at
pH 7.4 with of 0.0 to 20.0 equivalents of HH. Inset shows the fluorescence intensity at 458 nm vs. equiv.
of HH.
2.7. Binding mode and sensing mechanism of DIB
Based on the photophysical observations of DIB with HA and HH, we have proposed the mode
of interaction of the probe with the analytes as shown in Scheme 2. The formation of products C
1
and D (Scheme 2) was analyzed by H NMR experiment after separating the products from the
reaction of HH and HA, respectively with the probe DIB (Fig. 4). The NMR studies show that
the aldehydic proton (-CHO) signal at 10.03 ppm in DIB got disappeared and a new signal at
8.25 ppm is appeared for the azomethine proton (-CH=N-), which evidently indicates the
formation of condensation products C and D. Further, the imidazole-NH signal and aromatic
proton signals of the phenyl ring bearing aldehyde group show upfield shifts in products C and D
which signifies a decrease in electron delocalization in the products (C and D) in comparison to
probe DIB.
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OH
CH
NH
H
N
N
N
C
N
H
N
NH2
H
H2N
NH2-NH2.H2O
N
C
A
(Intermediate)
CHO
N
H
DIB
NH2-OH
OH
CH
H
N
N
N
C
N
H
N
OH
NH
HO
H
B
(Intermediate)
D
Scheme 2. A plausible reaction and sensing mechanism of probe DIB with hydroxyl amine (HA) and
hydrazine hydrate (HH)
This decrease in electron delocalization could be due to the chemical transformation from
aldehyde group in DIB to hydrazone and oxime units in C and D respectively, where the
carbonyl group possesses more electron withdrawing ability than the oxime and hydrazone units.
The FTIR spectroscopy also confirmed the formation of the condensation product D (Scheme 2)
resulting from the interaction of DIB with HA (Fig. S12). The band at 1695 cm-1 assigned to
>C=O stretching frequency of the aldehyde group in DIB get disappeared in product D
(DIB+HA). Besides, the appearance of a broad band near 3460 cm-1 and a sharp band at 1641
cm-1, respectively assigned to –OH and >C=N- stretching frequencies, clearly indicates the
formation of an oxime product D. The formation of products C and D was also confirmed from
the ESI-Mass experiments which showed a peak at m/z 338.93 and 339.93, respectively for the
hydrazone and oxime molecular ions (Fig. S13).
The fluorescence enhancement of DIB with HH and HA is probably because of the formation of
the condensation products C and D via the formation of the intermediate products A and B,
respectively. The electron withdrawing power of hydrazone and oxime units is comparatively
poorer in comparison to an aldehyde group. Thus, the overall electron delocalization from the
donor imidazole unit to the acceptor oxime group in product D is relatively less prominent than
that observed in probe DIB between the donor imidazole unit and acceptor aldehyde group as
confirmed from the NMR studies. The computational calculation also shows a stronger electron
delocalization in probe DIB than the products C and D (discussed in detail in section 2.8).
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Thus, when the probe (DIB) molecule is excited, it is expected to exhibit a stronger
intramolecular charge transfer (ICT) process in the excited state in comparison to the product D
that can lead to more non-radiative relaxation processes resulting in significant quenching of the
fluorescence signal.32 Further, the relatively poor solubility of the probe (DIB) in aqueous
solution in tandem with ICT process at the excited state might be responsible for its overall
decrease in the fluorescence signal. On the other hand, the introduction of –OH group in oxime
product D might enhances its solubility in aqueous medium together with a decrease in ICT
process at the excited state (due to poor electron withdrawing power of oxime group) could
possibly lead to the fluorescence enhancement of the probe (DIB) solution upon interaction with
HA.32
NH
-CH=N-
-CH=N-
(c)
(b)
(a)
NH
-CHO
NH
1
Fig. 4. H NMR spectra of (a) probe DIB, (b) product D (DIB+HA) and (c) product C (DIB+HH) in
DMSO-d6.
Moreover, the reason behind the fluorescence enhancement of DIB in presence of HH was
analyzed by dynamic light scattering (DLS) experiments to confirm whether the fluorescence is
due to the product C or as a consequence of molecular aggregation. It was observed that the
average particle size of DIB probe (50 µM) at 95% water is 182 nm and that of DIB+HH (50
µM each) is 199 nm, even after an incubation period of 5 hrs (Fig. S14). This result clearly
indicated that both DIB and its condensation product C (DIB + HH) have very similar
hydrodynamic radius (particle size). Thus, the enhancement in fluorescence properties of the
probe is possibly due to the formation of condensation product C, rather than any AIE effect. All
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these experimental findings clearly indicate that the formation of condensation products C and D
with HH and HA, respectively enhances the fluorescence signal of the probe DIB.
2.8. Computational studies
Now to understand the interaction of probe DIB with the analytes HA and HH, quantum
chemical calculations were performed on theoretical models by employing time-dependent
density functional theory (TD-DFT) using B3LYP functional and 6-31+G basis set in gas phase
and water solvent phase using Gaussian 09 program.33 The obtained optimized structures of
probe DIB, the intermediates A & B and the final products C & D (Scheme 2) were visualized
by using Gaussview 5 program and are shown in Fig. 5.
DIB
A
B
EW = -28092.7 eV
EW = -31135.7 eV
EW = -31675.6 eV
C
D
EW = -29056.9 eV
EW = -29597.0 eV
Fig. 5. Optimized structures of probe DIB, intermediates A & B and products C & D (colour key: white
= hydrogen; grey = carbon; blue = nitrogen; red = oxygen). EW indicates the calculated total energy in
water medium.
More information about the mechanism of interaction of DIB with the analytes was obtained
from the typical transition energy diagram for the highest occupied molecular orbital (HOMO)
and the lowest unoccupied molecular orbital (LUMO) of the probe along with its resultant
intermediates with hydroxyl amine and hydrazine as shown in Fig. 6. In probe DIB, the electron
distribution in HOMO is located over the entire molecule with a larger density on the imidazole
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and phenyl rings while in LUMO it is mainly concentrated over the benzaldehyde unit. On the
other hand, the electron distribution in HOMO and LUMO orbitals are located over the entire
molecule in intermediates A & B. Similarly, in the final products C & D, the electron density in
HOMO is distributed over the entire molecule while in LUMO it is largely spread over the
aromatic ring possessing the hydrazone (C) and oxime (D) groups (Fig. 6).
LUMO
LUMO
LUMO
LUMO
LUMO
-2
-3
-4
-5
-6
3.4 eV
3.15 eV
3.4 eV
3.17 eV
3.04 eV
A
B
C
D
DIB
HOMO
HOMO
HOMO
HOMO
HOMO
Fig. 6. The orbital shape and energy of the HOMO and LUMO of probe DIB and intermediates A & B
and products C & D calculated with DFT(B3LYP)/6-31+G method.
With reference to the energy of free probe DIB (-28092.7 eV), the optimized energies of the
intermediates A (-31135.7 eV), B (-31675.6 eV) and the products C (-29056.9 eV), D (-29597.0
eV) were found to be lower, which indicates the greater stability of the intermediates as well as
the products (Fig. 5). Further, these results indicate that the reaction of HA with the probe is
more favorable over HH as it forms a more stable intermediate B and product D in comparison to
A and C from HH. This observation is in good agreement with the time-dependent single
wavelength fluorescence studies which show the rapid formation of oxime over hydrazone with
probe DIB. The calculated energy of the HOMO and LUMO levels of the intermediates (A & B)
and products (C & D) were found to be increased from that of the free probe (Fig. 6) which can
be attributed due to the change in the electron distribution over the entire molecule by changing
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the nature of the functional group from aldehyde to hydrazone/oxime unit. Moreover, the HOMO
of probe DIB (-5.71 eV) is more stabilized over the HOMO of products C (-5.07 eV) and D (-
5.47 eV) that indicates a stronger electron delocalization in DIB in comparison to C and D which
is also confirmed from the proton NMR experiments.
3. Conclusions
In summary, we have synthesized a diphenyl imidazole based probe DIB through simple
condensation reactions in moderate yield. The probe DIB selectively detects hydroxyl amine
(HA) and hydrazine hydrate (HH) amongst various amines through a distinct visual fluorescence
change from non emissive to bright cyan and faint blue fluorescence respectively in 5% DMSO
phosphate buffer solution at pH 7.4. Interestingly, single wavelength time scan revealed that,
probe DIB could optically discriminate the presence of HA from HH through a more than 30
fold enhancement in fluorescence intensity and faster response toward HA. The proton NMR,
FTIR, ESI-Mass, DLS and DFT studies indicate that the enhancement in fluorescence signal of
the probe upon interaction with hydroxyl amine is due to the formation of oxime product which
modulates the ICT process in the excited state. The detection limit of DIB toward HA is found to
be 28 µM by fluorescence method indicating a greater sensitivity. Further the probe could detect
the HA over a pH range of 4.0 to 9.0 via a diagnostic cyan colour visual fluorescence response.
All together these results could pave the way toward the development of molecular indicators for
hydroxyl amine in multiple platforms.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
S.N.S gratefully acknowledges the financial assistance received from S&T Department, Govt. of
Odisha for the research grant. The research grant received from GNM Foundation for Prof. GN
Mahapatra Endowment Chair award is gratefully acknowledged. P. R and L. P are thankful to
School of Chemistry for providing necessary facilities to carry out this work. N. M is grateful to
S&T Department, Govt. of Odisha for a project fellowship. B.P.B is grateful to Department of
Biotechnology and Bioinformatics, Sambalpur University for providing necessary computational
facilities. Authors are thankful to Materials Research Centre, MNIT Jaipur, NMR facility of
Department of Chemistry, Ravenshaw University and ESI-Mass facility of NISER, Bhubaneswar
for recording the NMR and Mass spectra. A special thanks to Prof. Parameswar Krishnan Iyer
and Mr. Niranjan Meher of I.I.T Guwahati for carrying out DLS experiments, Dr. P. Parhi,
13

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Ravenshaw University, for carrying out NMR studies and Dr. C. S. PDuOrIo: 1h0i.1t0,39N/DI0SOBE0R06,08D
Bhubaneswar, for carrying out ESI-Mass studies reported in this paper. The financial assistance
received from UGC and DST, New Delhi under the DRS and FIST grant respectively to School
of Chemistry is gratefully acknowledged. We are thankful to Dr. H. Chakraborty, Sambalpur
University for his useful suggestions toward the improvement of this manuscript.
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