Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 201 (2018) 46–53

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular and Biomolecular

Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Exploration of solvent responsive Cr3+-Schiff base conjugates

formonitoring Cr3+ ions and organophosphates: Fabrication of
spot-testingdevices
Jaswant Singh a, Varinder Kaur a,⁎, Raghubir Singh b,⁎, Vimal K. Bhardwaj c
a
Department of Chemistry, Panjab University, Chandigarh 160014, India
b
Department of Chemistry, DAV College, Sector 10, Chandigarh 160011, India
c
Department of Chemistry, Indian Institute of Technology, Ropar 140001, India

a r t i c l e i n f o a b s t r a c t

Article history: Herein, we report 2-((2-hydroxybenzylidene)hydrazono)(phenyl)methyl)-5-methoxyphenol (SB) synthesized

Received 23 February 2018 by Schiff base condensation and characterized by spectroscopic techniques, elemental analysis and X-ray crystal-
Received in revised form 28 March 2018 lography. In solution phase, it interacts with Cr3+ ions and exhibits a prominent fluorimetric switch due to the
Accepted 26 April 2018
formation of SB·Cr3+ conjugate. The dual behavior of SB·Cr3+ conjugates i.e. self-aggregation in high water frac-
Available online 27 April 2018
tion (fw N 50%) and dissolution in low water fraction (fw b 50%) proves this conjugation excellent tool for moni-
Keywords:
toring Cr3+ ions. The SB·Cr3+ conjugate in methanol-water (70:30 v/v) allows quantification of Cr3+ ions with
Schiff base limit of detection 0.44 μM and its self-aggregation in high water fraction facilitates extraction of Cr3+ ions with
Fluorescence 95% extraction efficiency. Although SB interacts with Zn2+ ions which causes inference in the determination of
Cr3+ Cr3+ ions however the interferent can be easily masked with SCN− ions. Besides, the SB·Cr3+ conjugates are
Diethyl chlorophosphate also able to quantify organophosphate neurotoxins; i.e. diethyl chlorophosphate (with LOD 4.1 nM) and diethyl
Diethyl cyanophosphonate cyanophosphonate (with LOD 3.3 nM) from aqueous solutions. Moreover, SB and SB·Cr3+ conjugates can be
Self-aggregation coated on solid surfaces to fabricate portable devices for the on-spot detection of targets from real samples.
Hence, the conjugation of Schiff base and Cr3+ ions can be explored for the recognition, quantification and extrac-
tion of Cr3+ ions and detection of organophosphates.
© 2018 Published by Elsevier B.V.

1. Introduction analysis. Based on various advantages offered by Schiff bases, they

have been effectively explored for the detection and quantification of
Nowadays, molecules exhibiting self-aggregation are of immense in- metal ions [3–6,10–19]. Especially, aggregation with emission enhance-
terest due to their crucial role in hole-transport materials, ment properties of hydrazine Schiff base derivatives derived from 2-
spectrofluorimetric sensing and cell imaging [1–7]. Self-aggregation is hydroxynapthaldehyde and o-vanilin [10], and N, N′-
regulated by the chemical constitution and orientation of the substitu- diphenylbenzene-1,4-diamine and salicylaldehyde/2-
ents [2,8–10]. Especially, phenyl substituents in non-planar molecules hydroxynapthaldehyde have been exploited for the recognition of Zn2
restrict the intramolecular rotation and stimulate aggregation accompa- +
ions and F− ions, respectively in aqueous media [12].
nied with enhanced emission when solvent composition is varied Herein, we have derived a hydrazine based Schiff base (SB) which
[9,10]. This type of molecules exhibiting solvent responsive physico- forms conjugates with Cr3+ ions and displays a spectrofluorometric re-
chemical properties may act as probes with unique applicability in the sponse. The dissolved form of so formed SB·Cr3+ conjugates (in lower
routine analysis of various toxicants [11–13]. water fraction) has been utilized to standardize recognition and quanti-
Analytical strategies for monitoring a toxicant (i.e. recognition, fication protocols for monitoring Cr3+ ions and potentially hazardous
quantification and extraction) require effective interaction between organophosphates (i.e. diethyl chlorophosphate and diethyl
the probe and the target. For example; multi-donor Schiff base ligands cyanophosphonate). However, the aggregated form of SB·Cr3+ conju-
interact effectively with metal ions and serve as best probes for their gates in high percent water fraction has been explored for the extraction
of Cr3+ ions from aqueous systems. The dual nature of this conjugation
offered advantages of high sensitivity and selectivity in the recognition
⁎ Corresponding authors.
E-mail addresses: var_ka04@yahoo.co.in, (V. Kaur), raghu_chem2006@yahoo.com and quantification of Cr3+ ions as well as high precision and accuracy for
(R. Singh). their recovery to regenerate probe. To the best of our knowledge,

https://doi.org/10.1016/j.saa.2018.04.056
1386-1425/© 2018 Published by Elsevier B.V.
 J. Singh et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 201 (2018) 46–53 47

aggregation ability of a Schiff base has not been explored in the past for 2.2. Synthesis of Schiff Base (SB)
extraction purposes.
The targets (Cr3+ and organophosphates) investigated in the pres- In the first step, 2-hydroxy-4-methoxybenzophenone (20 mmol,
ent study are of high concern due to their widespread applications 4.6 g) and excess of hydrazine hydrate (100 mmol, 5.0 mL) were dis-
and toxicity issues. Chromium is mainly used for chromium plating, solved in methanol (50 mL). The mixture was stirred for 24 h at room
alloy manufacturing, and catalysis [20–22]; and diethyl temperature. A pale yellow solid product (HYD) was isolated by filtra-
chlorophosphate (DCP) and diethyl cyanophosphonate (DCNP) are the tion and washed thoroughly with methanol to remove unreacted hy-
potential chemical warfare agents [23–25]. The extensive usage of chro- drazine. In the second step, HYD (10 mmol, 2.4 g) and salicylaldehyde
mium in routine applications results in its bio-accumulation and as a (10 mmol, 1.1 mL) were dissolved in methanol (50 mL) by appropriate
consequence causes adverse effects on the living beings [26–28]. Simi- heating and then stirred for 10 h at ambient temperature. The solvent
larly, DCP and DCNP are malignant neurotoxins because they inhibit was evaporated under vacuum to afford a bright yellow solid. It was
proper functioning of acetylcholinesterase enzyme responsible for washed with diethyl ether to remove impurities and dried in a desicca-
nerve action [29]. Although, numerous chemosensors with conjugated tor. The crystals suitable for X-ray crystallography were obtained from
organic skeletons have been utilized for the spectrofluorometric recog- its methanolic solution by slow evaporation. Yield: 3.24 g, 94%. Melting
nition of Cr3+ ions (most commonly rhodamine, quinoline, point: 71 °C; C21H18N2O3 (346.39 g mol−1): Calcd, C 72.82, H 5.24, N
naphthyridine, thiazolothiazole, spirobenzopyran derivative, benzimid- 8.09, Found, C 72.52, H 5.14, N 8.03; IR (cm−1): 1614 (νC_N), 3036
azole derivative and Schiff base derivatives), however, their usage is (νH\\C_); 1H NMR (400 MHz, CDCl3): δ (ppm) 3.83 (s, 3H21), 6.35 (dd,
limited up to detection and/or quantification protocols [30–37]. In H19, 3J = 9.0, 4J = 2.52 Hz), 6.87 (m, H4), 6.89 (d, H20, 3J = 9.0 Hz),
order to extract the desired targets from various matrices, grafting of co- 6.57 (d, H17, 4J = 2.52 Hz), 6.98 (m, H2), 7.24–7.28 (m, H3,11,12,13),
ordinating systems on solid supports is required, which is laborious and 7.53 (m, H5), 7.54 (m, H10), 7.55 (m, H14), 8.69 (s, 1H, H7), 10.67 (s,
sometimes reduces the efficiency of encapsulating units [38,39]. In nut- 1H, HO-C16), 13.23 (s, 1H, HO-C1); 13C NMR (100 MHz, CDCl3): δ
shell, direct use of developed molecules performing all the functions (ppm) 55.5 (C21), 101.4 (C17), 107.0 (C19), 112.4 (C15), 117.2 (C2),
(i.e. detection, quantification and extraction) is limited for these targets. 117.8 (C6), 119.58 (C4), 127.8 (C11), 128.7 (C13), 129.3 (C10), 132.11
Hence, the conjugation between SB and Cr3+ can be considered as a (C14), 132.6 (C12), 132.9 (C20), 133.5 (C5), 133.9, (C3), 134.4 (C9),
multi-functional means for the recognition, quantification and removal 159.6 (C7), 161.9 (C8), 163.9 (C16), 164.7 (C1), 172.6 (C18); MS (m/z):
of the targets (Cr3+ and organophosphate). 347.07 [M + H]+.

2.3. Preliminary Photophysical Studies

2. Experimental
Firstly, SB was dissolved in methanol (0.125 mM) to record its UV–
2.1. Reagents Vis spectrum. Then, its solution (0.125 mM) in different solvents (i.e.
chloroform, tetrahydrofuran, dimethylsulphoxide, dichloromethane,
All the reagents such as 2-hydroxy-4-methoxybenzophenone (Al- isopropanol, isobutanol, dimethylformamide, methanol and water)
drich), hydrazine (Qualigens™), salicylaldehyde (Acros), copper(II) ni- was excited at 420 nm to scrutinize respective emission profile. To
trate trihydrate (HPLC™, India), cobalt(II) nitrate (Merck, India), audit the behavior of SB towards different metallic species, 2.0 mL SB so-
nickel(II) nitrate (Merck, India), iron(III) chloride (Merck, India), alu- lution (0.125 mM in methanol) was added with 10 μL metal salt solution
minium(III) chloride (Merck, India), iron(II) sulphate (Merck, India), (i.e. Cr3+, Hg2+, Al3+, Fe2+, Fe3+, Zn2+, Cd2+, Co2+, Ni2+, Cu2+, Mn2+
zinc(II) nitrate hexahydrate (HPLC™, India), cadmium(II) nitrate respectively; 1.0 mM each) and emission spectrum was recorded (λexc
(Merck, India), chromium(III) nitrate (Merck, India), lead(II) nitrate 420 nm) for each solution.
(Merck, India), mercury(II) chloride (Merck, India), ammonium thiocy-
anate (Loba Chime, India), sodium sulphate (Loba Chemie, India), so- 2.4. Solvent Responsive Self-aggregation of SB-Cr3+ Conjugate
dium disulphate (Nice, India), sodium periodate (Nice, India), sodium
nitride (Loba Chemie, India), sodium formate (Loba Chime, India), so- Briefly, 2.0 mL SB solution (0.125 mM, in methanol) was taken in dif-
dium carbonate (Finar, India), sodium bromate (Finar, India), sodium ferent test tubes and Cr3+ solution (0.3 mL of 0.75 mM in methanol)
tetrafluoroborate (Spectrochem, India), potassium peroxydisulphate was added in each to make 1:1 stoichiometry. The total volume of
(Nice, India), sodium benzoate (Loba Chemie, India), triethyl phosphite each solution was made 5 mL with varying solvent composition i.e.
(Aldrich), diethyl chlorophosphate (TCI-India), diethyl methanol-water in 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30,
cyanophosphonate (Aldrich), tetraisopropyl methylene diphosphonate 80:20 and 90:10 ratios, respectively. The solutions were shaken thor-
(TCI-India), diethyl ethylphosphonate (Aldrich), trimethyl phosphate oughly and fluorescence spectrum was recorded for each solution. To
(Aldrich) and dichlorvos (Sigma) were purchased commercially and know the particle size of the aggregates, dynamic light scattering exper-
used as such without further purification. iments were done using clean optically translucent disposable plastic
The organic solvents such as methanol and diethyl ether were pur- cuvettes. For this, different samples of SB·Cr3+ aggregates (0.05 mM
chased from Finar (India) and purified by the reported methods [40]. each) were prepared in water-methanol solvent mixture viz. 10–90%
Silica gel 60 F254 coated TLC aluminium sheets (Merck) were used for v/v water fraction and 1.0 mL of each solution was taken for particle
real time analysis. Stock solutions such as 100 mL SB solution in meth- size analysis. The data was recorded for each solution and a graph for in-
anol/water, 7:3 v/v (0.125 mM), 50 mL chromium(III) nitrate in metha- tensity versus particle size (r. nm) was plotted.
nol/water, 7:3 v/v (0.75 mM), 50 mL zinc(II) nitrate hexahydrate in
water (0.75 mM), 10 mL EDTA solution in water (0.25 mM), 10 mL 2.5. Quantification of Cr3+ Ions
diethyl chlorophosphate solution in water (100 mM), 10 mL diethyl
cyanophosphonate solution in water (100 mM) were prepared and Before quantification, formation and stability of SB-Cr3+ conjugates
stored in closed vessels below 4 °C. Due to persistent poisoning, the so- in solution phase was investigated by performing some preliminary ex-
lutions of organophosphates were prepared in fume-hood under all periments like pH optimization, Job plot, and interference due to anions
safety measures such as use of gloves, eyeglasses and masks. After com- and cations, and removal of interference (details given in ESI). For the
pletion of quantification, standard protocol was followed for the dis- quantification of Cr3+ ions, 10 μL of potassium hydrogen sulphate buffer
posal of organophosphates. The details of instrumentation used is was added to SB solution (0.125 mM in methanol/water, 7:3 v/v) to
given in ESI. maintain the pH value near 5.5. A spectrofluorimetric titration was
48 J. Singh et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 201 (2018) 46–53

performed by incremental addition of 10 μL of Cr3+ ions (0.75 mM in Furthermore, SB and SB·Cr3+ conjugates were utilized for the fabri-
methanol/water, 7:3 v/v) and response was recorded after each addi- cation of spot testing device and method was validated by determining
tion. The data was plotted to obtain calibration curve, limit of detection, some real and synthetic samples (see experimental details in ESI).
linear range and limit of quantification.
3. Results and Discussion
2.6. Extraction of Cr3+ Ions
3.1. Synthetic, Structural and Photophysical Aspects of SB
3+
2.0 mL SB (0.125 mM) solution was taken in a test tube and Cr so-
lution (0.3 mL of 0.75 mM) was added in 1:1 stoichiometry to get A hydrazine-based Schiff base (SB) with ONNO donor set was syn-
SB·Cr3+ conjugate. The total volume was made 5.0 mL by the addition thesized by following a two-step procedure reported for similar type
of water for significant aggregation. The aggregates formed (SBAG·Cr3 of molecules [10]. In the first step, a hydrazone derivative (HYD) was
+
) were kept undisturbed (up to 6 h) for clear visual separation. The obtained by the reaction of 2-hydroxy-4-methoxybenzophenone and
upper layer was decanted and aggregates were isolated for the recovery hydrazine, which was reacted with salicylaldehyde in the second step
of Cr3+ ions. to obtain Schiff base (SB) (Scheme 1).
To know the extraction efficiency, 10 μL solution was withdrawn The formation of SB was confirmed by IR, NMR (1H, 13C), ESI-MS, el-
from the upper layer, added with 2.0 mL of SB (0.125 mM) and used emental analysis and single crystal X-ray diffraction. The solid-state IR
to know the final concentration of metal ions. The difference between spectrum of SB exhibited a band at 1614 cm−1 pertaining to\\C_N\\
initial and final concentration of metal ions in the solution was used to vibration confirming the formation of azomethinic bond. It was also
calculate Cr3+ ions extracted in the form of SBAG·Cr3+. The procedure supported by 1H NMR, which showed a sharp signal at 8.69 ppm for
was repeated ten times to check the reproducibility of the results. azomethinic proton (Fig. 1). In addition, the signals at 10.67 and
13.23 ppm suggested the presence of intramolecularly hydrogen
bonded\\OH groups, which was also supported by crystal structure of
2.7. Recovery of Cr3+ Ions and Reusability of SB SB. The 1H and 13C NMR signals pertaining to aromatic region were
well resolved. The ESI-MS analysis showed a base peak at m/e 347.02
To recover Cr3+ ions and check the reusability of SB, SBAG·Cr3+ ag- corresponding to [M + H] + ion (Fig. 2).
gregates (discussed in Section 2.5) were dissolved in 10 mL methanol/ The single crystal X-ray diffraction patterns of SB revealed mono-
water (7:3; v/v) mixture. 2.0 mL of this solution was titrated with clinic system with space group P21/c (Fig. 3). The important crystallo-
0.25 mM EDTA solution with the incremental addition (of 10 μL) and graphic information is listed in Table 1. The non-planarity of the
emission was recorded at each step. The process was followed by com- molecule was justified by interplanar angle of 119.47° between rings
plete quenching of band at 470 nm was achieved. The free ligand was R1 and R2, 148.39° between R1 and R3, and 98.84° between R2 and
collected as aggregate by increasing water fraction. The method was re- R3. The non-planar structure of SB introduced self-aggregation and as
peated ten times to check the reproducibility of results. a consequence aggregation induced emission enhancement (AIEE) in
the molecule. In SB, phenyl substituents are non-planar and restrict
2.8. Quantification of Organophosphates (OPs) the intramolecular rotation. From literature review, this type of behav-
ior stimulate aggregation with enhanced emission [3,10].
The SB·Cr3+ conjugate solution was utilized for the detection and The absorption and emission spectra of SB recorded in methanol ex-
quantification of OPs in water. Firstly, 2.0 mL of SB (0.125 mM in meth- hibited an absorption band centered at 380 nm and a very weak emis-
anol/water; 7:3 v/v) and 30 μL of Cr3+ ion solution (0.75 mM in metha- sion band centered at 550 nm when excited at 420 nm. A similar
nol/water; 7:3 v/v) were taken in a cuvette to obtain SB·Cr3+ conjugate. trend was observed for emission profile in other solvents except water
To the same solution, 10 μL of different OPs (100 mM in water each of in which a sharp enhancement in the emission band was observed
triethyl phosphite, diethyl chlorophosphate, diethyl cyanophosphonate, (Fig. S2). Different spectrofluorometric behavior in water may be attrib-
tetraisopropyl methylene phosphonate, diethyl ethylphosphonate, uted to the phenomenon of aggregation induced emission enhance-
trimethyl phosphate and dichlorvos) were added and the emission ment because SB formed aggregates in pure water. The
was recorded at 470 nm. spectrofluorometric response of SB (0.125 mM) in the presence of dif-
For the quantification of diethylchlorophosphate, a ferent bivalent and trivalent metal ions (such as Fe2+, Fe3+, Cu2+, Cr3
spectrofluorimetric titration of SB·Cr3+ conjugate solution was done +
, V3+, Al3+, Hg2+, Zn2+, Cd2+, Pb2+, Co3+, Ni2+ and Mn2+ ions) re-
by the incremental addition of diethyl chlorophosphate (100 mM in vealed origin of a new emission band centered at 470 nm (λexc =
water). The emission was documented after each addition and similar 420 nm) in the presence of Cr3+ ions. Besides, a relatively weak spectro-
procedure was followed with diethyl cyanophosphonate (100 mM in fluorometric response for Zn2+ ions was also observed with the origin
water). All the parameters such as calibration curve, linear range, limit of emission band centered at 505 nm (Fig. S3). This response of SB to-
of detection, and limit of quantification were determined from the re- wards Cr3+ and Zn2+ ions can be attributed to selective chelation en-
corded data. hanced fluorescence effect (CHEF) [41]. Due to strong response in case

Scheme 1. Synthetic route for the formation of SB from 2-hydroxy-4-methoxybenzophenone, hydrazine and salicylaldehyde via hydrazone (HYD) formation (numbering of carbon atoms
is given to assign 1H and 13C NMR signals, R1, R2 and R3 are given to indicate rings).
 J. Singh et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 201 (2018) 46–53 49

Fig. 1. 1H NMR spectrum and (above) and 13C NMR spectrum of SB in CDCl3 (below).

of chromium (relative to Zn2+ ions), SB was further explored for its

monitoring and parameters were optimized to mask the interference
of zinc.

Fig. 3. ORTEP view of SB with all non hydrogen atoms drawn at 20% probability and
hydrogen atoms at 2% probability (R1, R2 and R3 are assigned to give interplanar angle).
Fig. 2. Mass spectrum of SB. The bond lengths and bond angles are given in Table S1.
50 J. Singh et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 201 (2018) 46–53

Table 1
Parameters of X-ray diffraction data collection and structure refinement of SB.

Parameter Data Parameter Data

Molecular formula C21H18N2O3 Absorption coefficient (mm−1) 0.088

Molecular weight 346.39 F (000) 736
Temperature (K) 273(2) K Crystal size (mm3) 0.1 × 0.1 × 0.1
Wavelength (Å) 0.71073 Theta range for data collection 2.643 to 25.499°
Crystal system Monoclinic Index ranges −9 ≤ h ≤ 9, −33 ≤ k ≤ 33, −9 ≤ l ≤ 9
Space group P 21/c Reflections collected 25,964
Unit cell dimensions a = 8.0171(4) Å Independent reflections [R(int) = 0.0511] 3308
b = 27.9196(15) Å Completeness to theta = 25.242° 99.8%
c = 7.9422(4) Å Refinement method Full-matrix least-squares on F2
α = 90° Data/restraints/parameters 3308/0/243
β = 90.749 (2) ° Goodness-of-fit on F2 1.052
γ = 90° Final R indices [I N 2sigma(I)] R1 = 0.0576, wR2 = 0.1372
Volume (Å3) 1777.58(16) R indices (all data) R1 = 0.0914, wR2 = 0.1587
Z 36 Largest diff. peak and hole (e·Å−3) 0.137 and −0.464
Density (calc.) (mg/m3) 1.302

3.2. Solvent Responsive Self-aggregation of SB·Cr3+ Conjugate 3.3.1. Quantification of Cr3+ Ions
As discussed previously, SB experienced a fluorimetric switch in the
SB·Cr3+ conjugate showed complete dissolution in methanol and presence of Cr3+ ions and an emission band centered at 470 nm was ob-
produced an emission band centered at 470 nm. In aqueous system, it served due to the formation of SB·Cr3+ conjugates. The response of SB
exhibited self-aggregation and aggregation induced emission enhance- towards Cr3+ ions was best in weakly acidic conditions (pH 5.5–6.0)
ment (AIEE), which increased considerably with increasing water frac- which might be due to maximum conjugation between SB and Cr3+
tion (fw N 40% v/v) in binary solvent system (methanol-water). The ions via donor-acceptor interaction (Fig. S6). The conjugates were
aggregation could be seen clearly by naked eye and in UV light found unstable in highly acidic media due to the blockage of donor
(shown in Fig. S4). The emission enhancement also increased with sites by proton or decomposition of conjugates. Similarly, in basic
time and attained a constant plateau after 10 min suggesting stabiliza- media deprotonation of SB increases the self-association and decreases
tion of aggregates (Fig. 4). Dynamic light scattering measurements per- conjugation of Cr3+ ions. The Job plot revealed 1:1 metal-ligand stoichi-
formed for insight investigation of SB·Cr3+ conjugates revealed average ometry of the SB·Cr3+ conjugates (Fig. S7). The continuous addition of
particle size of ca. 67 nm in methanol-water (9:1 v/v). However, upon Cr3+ solution (0.75 mM in methanol/water 7:3 v/v) to SB demonstrated
incremental addition of water (from 10% to 90%), the average size of a linear enhancement of the band at 470 nm from 0.75 to 98 μM (Fig. 5).
SBAG·Cr3+ was increased attaining a particle size of ca. 1372 nm. This The quantification parameters i.e. regression equation, limit of detection
observation suggested the significant aggregation of SB·Cr3+ in higher (using 3σ/k) and limit of quantification (using 10σ/k) for Cr3+ ions
water fraction (fw b 50) (Fig. S5). were determined to be 3.3425 × 107 [Cr3+] + 1267.73 (R2 = 0.99),
0.44 μM, and 1.46 μM, respectively.
The interference studies revealed negligible effect of anions (up to
3.3. Analysis of Cr3+ Ions five equivalents) and cations (up to ten equivalents) except Zn2+ ions
on the quantification of Cr3+ ions (Fig. S8). Therefore, in order to re-
The dissolution of SB at fw ≤ 30% and aggregation at fw ≥ 30% made it a move the interference of Zn2+ ions completely, sodium thiocyanate so-
useful means for recognition and extraction of Cr3+ ions by simply vary- lution was used. The effect of SCN− was studied in two parallel
ing solvent system. Herein, methanol-water (70:30 v/v) was used to experiments. In the first experiment, emission spectrum of SB·Zn2+
standardize a method for Cr3+ ion recognition/quantification and was monitored with the incremental addition of sodium thiocyanate.
methanol-water (30:70 v/v) was used for the extraction purpose. The emission band centered at 505 nm due to SB·Zn2+ conjugate

Fig. 4. Emission profile of SB·Cr3+ (λexc 420 nm) in varied water-methanol composition (viz.10–90% v/v); Inset: Time optimization for aggregation of same solutions.
 J. Singh et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 201 (2018) 46–53 51

Fig. 5. Spectrofluorometric titration of SB against Cr3+ ions (with the incremental addition
of 10 μL) at an excitation of 420 nm; Inset: calibration curve at 470 nm with increasing
concentration of Cr3+ ions in SB solution (in methanol-water; 7:3 v/v). Fig. 6. Quenching of SB·Cr3+ signal (at 470 nm) in the presence of different
organophosphates (DEEP-diethyl ethylphosphonate; DCNP-diethyl cyanophosphonate;
TEEP-triethyl phosphite; TMEP-tetraisopropyl methylene diphosphonate; TMP-
trimethyl phosphate; DCP-diethyl chlorophosphate; DCV-dichlorvos).
diminished continuously suggesting extraction of Zn2+ ions leaving the
SB probe free (Fig. S9). In the parallel experiment, emission studies of
mixture of Cr3+ and Zn2+ was investigated with the incremental addi- regression equation and limit of detection for DCP were −3.62 × 109
tion of sodium thiocyanate. Initially, the mixture of metal ions possessed [DCP] + 13,503.61 (R2 = 0.98) and 4.1 nM, respectively (Fig. S11). Sim-
a merged emission signal centered at 484 nm. However, this band ilarly, the respective values for DCNP were −3.90 × 109 [DCNP]
slowly shifted to 470 nm with the addition of sodium thiocyanate and + 15,643.8 (R2 = 0.98) and 3.3 nM.
attained a constant plateau indicating the removal of interference
from the spectrofluorometric response. The masking of Zn2+ with 3.6. Fabrication of Spot Testing Devices
SCN− ions is also reported in literature [42]. Hence, sodium thiocyanate
can be used as a masking agent to detect and quantify Cr3+ ions selec- To apply the probe for routine analysis, firstly SB@SiO2@Al strips
tively in the presence of Zn2+ ions. were prepared by dipping TLC aluminium strips in methanolic solution
of SB (See experimental details in ESI). The strips (Fig. 7a) became fluo-
3.3.2. Extraction of Cr3+ Ions rescent when dipped in the aqueous solutions of chromium nitrate
As discussed earlier, aggregation of SB·Cr3+ conjugate was observed (Fig. 7b). However, the fluorescence of the strips lost when interacted
above fw ≥ 50%. Therefore, this particular behavior was explored to ex- with DCP and DCNP in aqueous media (Fig. 7c, d). The observations on
tract Cr3+ ions from the samples. The extraction method works on en- TLC strips were consistent with the detection of metal ions and organo-
capsulation of Cr3+ ions in organic media as SB·Cr3+ conjugate phosphates in solution phase.
followed by the addition of excessive water to form SBAG·Cr3+ aggre- Validation of standardized protocol for real time analysis (in solution
gates. The amount of aggregated conjugate was calculated to be ca. phase) was done using industrial scrap, pharmaceutical samples, soil
95%. The reproducibility of method was confirmed by repeating the ex- samples collected from construction sites and water samples collected
periments ten times which presented a relative standard deviation of from different sources (experimental details in ESI). The results listed
0.24% (Table S2). in Table 2 showed the excellent applicability of optimized procedures.

3.4. Regeneration of SB and Recovery of Cr3+ Ions

A crucial requirement for an extractant is its regeneration and this

was achieved successfully in the present case by treating SBAG·Cr3+
with EDTA solution. The spectrofluorimetric investigation of solution
obtained by the dissolution of aggregates revealed diminishing of signal
for SB·Cr3+ (at 470 nm), with the incremental addition of EDTA and si-
multaneous regeneration of signal at 550 nm (due to SB). This con-
firmed the regeneration of SB and recovery of Cr3+ ions a EDTA
chelates (Fig. S10). The regenerated SB was separated as aggregate
from the solution by increasing the water content.

3.5. Quantification of Organophosphates

During the interference studies, a significant quenching of SB·Cr3+

signal was observed in the presence of phosphates. This aspect moti-
vated us to use the SB·Cr3+ for the quantification of organophosphates.
Out of various organophosphates tested in aqueous medium (see exper-
imental details in ESI), diethyl chlorophosphate and diethyl
Fig. 7. (a) SB@SiO2@Al strips (b) SB@SiO2@Al strips after interaction with Cr3+ ions (in
cyanophosphonate showed significant quenching of emission band of methanol/H2O; 7:3 v/v), (c) Cr3+·SB@SiO2@Al strips after interaction with aqueous
SB·Cr3+ conjugate (at 470 nm) (Fig. 6). The quantification parameters solution of DCP (d) Cr3+·SB@SiO2@Al strips after interaction with aqueous solution of
were obtained by titrating SB·Cr3+ against organophosphates. The DCNP.
52 J. Singh et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 201 (2018) 46–53

Table 2
Real sample analysis using SB probe.

S. no. Source Sample type/material Metal/OPs Accuracy ± RSD (%)

1 Industrial scrap Chromium electroplated wires of bicycle rim 0.178 mM (Cr3+) –

2 Pharmaceuticals (multi-vitamin tablet) Methylcobalamin: 1500 μg Cr: 470 μg 94.0 ± 0.24
Folic acid IP: 1.5 mg
Alpha lipoic acid: 100 mg
Vitamin B6 IP: 1.5 mg
Chromium picolinate: 500 μg
ZnSO4·H2O IP 7.5 mg
3 Construction site Spiked soil (DCP, 15 μM; DCNP, 15 μM) 13.4 μM (DCP) 89.3 ± 1.55
14.1 μM (DCNP) 94.0 ± 0.73
4 Tap water Spiked water sample (DCP, 15 μM; DCNP, 15 μM) 14.8 μM (DCP) 98.6 ± 0.13
14.2 μM (DCNP) 94.6 ± 0.12
5 Lake water Spiked water sample (DCP, 15 μM; DCNP, 15 μM) 13.7 μM (DCP) 91.3 ± 0.09
12.4 μM (DCNP) 82.7 ± 0.01

The amount of Cr3+ ions present in the samples was calculated from re- conjugation of Cr3+ and SB can be used for selective recognition, highly
gression equation. The accuracy of results was found to be 82–94%. A t- sensitive quantification and efficient extraction of the target. Moreover,
test was performed on the data set collected for a test sample (by it can be used for the fabrication devices for on-spot recognition of the
performing 15 experiments), which revealed a systematic error in the targets.
data set. The relative standard deviation was calculated for each data
set. The quantification of DCP and DCNP was done by determining the 4. Conclusions
spiked samples of river water and tap water. The spiked DCP and
DCNP samples were quantified with 98% accuracy (Table 2). Herein, a hydrazine-based Schiff base probe SB has been developed
for the recognition, quantification and extraction of Cr3+ ions. The SB
3.7. Comparison With Existing Methods displays a quick and selective fluorimetric switch ‘ON’ response due to
the formation of 1:1 SB·Cr3+ conjugate and can extract about 95% of
The important features and working conditions of the developed the metal ions by self-aggregation (as SBAG-Cr3+) in high water fraction.
probe are summarized in Fig. 8. These properties proved SB better The SB probe can be recovered from the aggregates by the addition of
over the existing Schiff base probes due to their weaknesses like; utiliza- EDTA solution. Moreover, Zn2+ ions can be masked by thiocyanate
tion of highly toxic solvents, non-recovery of probe, suitability for single ions and the Cr3+ ions can be quantified and extracted selectively. SB-
application (either recognition/quantification or extraction) and higher Cr3+ have been further used for the detection and quantification of po-
detection limits (details are given in Table. S3) [30,34–37,43,44]. tentially neurotoxic organophosphates from environmental samples.
In contrast, the protocol reported herein is based on the use of mixed The strategies have been optimized and applied for the quantification
organic-aqueous solvent systems and can be regenerated for reuse. It af- of metallic species and OPs in real samples.
fords advantage of recovery of the target using EDTA. Further, the

Acknowledgements

Authors are grateful to UGC, New Delhi for Meritorious fellowship

vide no. F.25.1/2014(BSR)/No.5-5-91/2007(BSR), CSIR, New Delhi
(sanction no. 01(2909)/17/EMR-II) and DST-FIST for providing financial
support vide no. SR/FST/college-335/2016.

Appendix A. Supplementary Data

CCDC 1564371 contains the supplementary crystallographic data for

SB. The data can be obtained free of charge via http://www.ccdc.cam.ac.
uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-
033; or e-mail: deposit@ccdc.cam.ac.uk. Supplementary data to this ar-
ticle can be found online at https://doi.org/10.1016/j.saa.2018.04.056.

References
[1] R.T.K. Kwok, C.W.T. Leung, J.W.Y. Lam, B.Z. Tang, Biosensing by luminogens with
aggregation-induced emission characteristics, Chem. Soc. Rev. 44 (2015)
4228–4238, https://doi.org/10.1039/c4cs00325j.
[2] Y. Hong, W.Y. Lam, B. Zhong, Aggregation-induced emission: phenomenon, mecha-
nism and applications, Chem. Commun. (2009) 4332–4353, https://doi.org/10.
1039/b904665h.
[3] B. Liu, H. Zhou, B. Yang, X. Hu, Aggregation–induced emission activity and further
Cu2+ induced self-assembly process of two Schiff compounds, Sens. Actuator B-
Chem. 246 (2017) 554–562, https://doi.org/10.1016/j.snb.2017.02.120.
[4] I. Kaur, P. Kaur, K. Singh, 2-(4-Amino-2-hydroxyphenyl)benzothiazole based Schiff-
base: complexation/decomplexation driven photo physical tuning of fluorescence
leading to Cu2+ and PO3− 4 detection, Sens. Actuator B-Chem. 257 (2018)
1083–1092, https://doi.org/10.1016/j.snb.2017.10.173.
Fig. 8. Illustration of important features and working conditions of the SB probe for the [5] P. Singh, H. Singh, V. Vanita, R. Sharma, G. Bhargava, S. Kumar, Nanomolar Cu2+ de-
detection, quantification and extraction of Cr3+ and OPs (DCP and DCNP). tection in dater based on disassembly of AIEgen: applications in blood serum, cell
 J. Singh et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 201 (2018) 46–53 53

imaging and complex logic circuits, Chem. Sel. 1 (2016) 6880–6887, https://doi.org/ [25] S. Hassani, S. Momtaz, F. Vakhshiteh, A.S. Maghsoudi, M.R. Ganjali, P. Norouzi, M.
10.1002/slct.201601497. Abdollahi, Biosensors and their applications in detection of organophosphorus pes-
[6] U. Panda, S. Roy, D. Mallick, A. Layek, P.P. Ray, C. Sinha, Aggregation induced emis- ticides in the environment, Arch. Toxicol. 91 (2017) 109–130, https://doi.org/10.
sion enhancement (AIEE) of fluorenyl appended Schiff base: a turn on fluorescent 1007/s00204-016-1875-8.
probe for Al3+, and its photovoltaic effect, J. Lumin. 181 (2017) 56–62, https://doi. [26] D. Marmolino, Friedreich's ataxia: past, present and future, Brain Res. Rev. 67 (2011)
org/10.1016/j.jlumin.2016.07.008. 311–330, https://doi.org/10.1016/j.brainresrev.2011.04.001.
[7] L. Ravotto, P. Ceroni, Aggregation induced phosphorescence of metal complexes: [27] K.J. Barnham, A.I. Bush, Metals in Alzheimer's and Parkinson's diseases, Curr. Opin.
from principles to applications, Coord. Chem. Rev. 346 (2017) 62–76, https://doi. Chem. Biol. 12 (2008) 222–228, https://doi.org/10.1016/j.cbpa.2008.02.019.
org/10.1016/j.ccr.2017.01.006. [28] A.K. Shanker, C. Cervantes, H. Loza-Tavera, S. Avudainayagam, Chromium toxicity in
[8] B. Zhong, Z. Zhao, S. Chen, X. Shen, F. Mahtab, Y. Yu, P. Lu, J.W.Y. Lam, S. Kwok, B. plants, Environ. Int. 31 (2005) 739–753, https://doi.org/10.1016/j.envint.2005.02.
Zhong, Aggregation-induced emission, self-assembly and electroluminescence, 003.
Chem. Commun. 46 (2010) 686–688, https://doi.org/10.1039/b915271g. [29] Y. Chen, Organophosphate-induced brain damage: mechanisms, neuropsychiatric
[9] Y. Hong, W.Y. Lam, B. Zhong, Aggregation-induced emission, Chem. Soc. Rev. 40 and neurological consequences, and potential therapeutic strategies,
(2011) 5361–5388, https://doi.org/10.1039/c1cs15113d. Neurotoxicology 33 (2012) 391–400, https://doi.org/10.1016/j.neuro.2012.03.011.
[10] M. Shyamal, P. Mazumdar, S. Maity, S. Samanta, G.P. Sahoo, A. Misra, Highly selective [30] J. Wang, Q. Qi, L. Zhang, S. Li, Turn-on luminescent sensing of metal cations via
turn-on fluorogenic chemosensor for robust quantification of Zn(II) based on aggre- quencher displacement: rational design of a highly selective chemosensor for chro-
gation induced emission enhancement feature, ACS Sensors 1 (2016) 739–747, mium(III), Inorg. Chem. 51 (2012) 13103–13107, https://doi.org/10.1021/
https://doi.org/10.1021/acssensors.6b00289. ic3009267.
[11] M. Shellaiah, Y.-H. Wu, A. Singh, M.V. Ramakrishnam Raju, H.-C. Lin, Novel pyrene- [31] S.Y. Lee, K.H. Bok, J.A. Kim, S.Y. Kim, C. Kim, Simultaneous detection of Cu2+ and Cr3
and anthracene-based Schiff base derivatives as Cu2+ and Fe3+ fluorescence turn- +
by a simple Schiff-base colorimetric chemosensor bearing NBD (7-nitrobenzo-2-
on sensors and for aggregation induced emissions, J. Mater. Chem. A 1 (2013) oxa-1,3-diazolyl) and julolidine moieties, Tetrahedron 72 (2016) 5563–5570,
1310–1318, https://doi.org/10.1039/C2TA00574C. https://doi.org/10.1016/j.tet.2016.07.051.
[12] Y. Xie, G. Shan, P. Li, Z. Zhou, Z. Su, A novel class of Zn(II) Schiff base complexes with [32] M. Sarkar, S. Banthia, A. Samanta, A highly selective “off–on” fluorescence
aggregation-induced emission enhancement (AIEE) properties: synthesis, charac- chemosensor for Cr(III), Tetrahedron Lett. 47 (2006) 7575–7578, https://doi.org/
terization and photophysical/electrochemical properties, Dyes Pigments 96 (2013) 10.1016/j.tetlet.2006.08.091.
467–474, https://doi.org/10.1016/j.dyepig.2012.09.020. [33] Y.R. Bhorge, H.T. Tsai, K.F. Huang, A.J. Pape, S.N. Janaki, Y.P. Yen, A new pyrene-based
[13] T. Simon, M. Shellaiah, V. Srinivasadesikan, C. Lin, F. Ko, K.W. Sun, M. Lin, A simple Schiff-base: a selective colorimetric and fluorescent chemosensor for detection of Cu
pyrene based AIEE active Schiff base probe for selective naked eye and fluorescence (II) and Fe(III), Spectrochim. Acta Mol. Biomol. Spectrosc. 130 (2014) 7–12, https://
off-on detection of trivalent cations with live cell application, Sens. Actuator B- doi.org/10.1016/j.saa.2014.03.110.
Chem. 231 (2016) 18–29, https://doi.org/10.1016/j.snb.2016.02.136. [34] Y. Joon, Y. Han, H. Young, J. Young, I. Hong, C. Kim, A colorimetric and fluorescent
[14] J. Yan, L. Fan, J.C. Qin, C.R. Li, Z.Y. Yang, A novel and resumable Schiff-base fluorescent chemosensor for selective detection of Cr3+ and Al3+, Inorg. Chem. Commun. 33
chemosensor for Zn(II), Tetrahedron Lett. 57 (2016) 2910–2914, https://doi.org/10. (2013) 48–51, https://doi.org/10.1016/j.inoche.2013.04.017.
1016/j.tetlet.2016.05.079. [35] Z. Li, W. Zhao, Y. Zhang, L. Zhang, M. Yu, J. Liu, H. Zhang, An off-on fluorescent
[15] W. Wang, R. Li, T. Song, C. Zhang, Y. Zhao, Study on the fluorescent chemosensors chemosensor of selectivity to Cr3+ and its application to MCF-7 cells, Tetrahedron
based on a series of bis-Schiff bases for the detection of zinc(II), Spectrochim. Acta 67 (2011) 7096–7100, https://doi.org/10.1016/j.tet.2011.07.008.
Mol. Biomol. Spectrosc. 164 (2016) 133–138, https://doi.org/10.1016/j.saa.2016. [36] Y. Wu, C. Li, Y. Li, J. Tang, D. Liu, A ratiometric fluorescent chemosensor for Cr3+
04.016. based on monomer-excimer conversion of a pyrene compound, Sens. Actuator B-
[16] W. Al Zoubi, N. Al Mohanna, Membrane sensors based on Schiff bases as chelating Chem. 203 (2014) 712–718, https://doi.org/10.1016/j.snb.2014.07.046.
ionophores-a review, Spectrochim. Acta Mol. Biomol. Spectrosc. 132 (2014) [37] P.S. Nayab, M. Shkir, Rapid and simultaneous detection of Cr(III) and Fe(III) ions by a
854–870, https://doi.org/10.1016/j.saa.2014.04.176. new naked eye and fluorescent probe and its application in real samples, Sens Actu-
[17] W. Fang, G. Zhang, J. Chen, L. Kong, L. Yang, H. Bi, J. Yang, An AIE active probe for spe- ators B-Chem. 251 (2017) 951–957, https://doi.org/10.1016/j.snb.2017.05.102.
cific sensing of Hg2+ based on linear conjugated bis-Schiff base, Sens. Actuator B- [38] P.K. Jal, S. Patel, B.K. Mishra, Chemical modification of silica surface by immobiliza-
Chem. 229 (2016) 338–346, https://doi.org/10.1016/j.snb.2016.01.130. tion of functional groups for extractive concentration of metal ions, Talanta 62
[18] X. Cao, X. Zeng, L. Mu, Y. Chen, R.X. Wang, Y.Q. Zhang, J.X. Zhang, G. Wei, Character- (2004) 1005–1028, https://doi.org/10.1016/j.talanta.2003.10.028.
ization of the aggregation-induced enhanced emission, sensing, and logic gate be- [39] I. López-García, M.J. Muñoz-Sandoval, M. Hernández-Córdoba, Cloud point
havior of 2-(1-hydroxy-2-naphthyl)methylene hydrazone, Sens. Actuator B-Chem. microextraction involving graphene oxide for the speciation of very low amounts
177 (2013) 493–499, https://doi.org/10.1016/j.snb.2012.11.003. of chromium in waters, Talanta 172 (2017) 8–14, https://doi.org/10.1016/j.talanta.
[19] D. Xie, Z. Ran, Z. Jin, X. Zhang, D. An, Simple fluorescent probe for Zn(II) based on the 2017.05.017.
aggregation-induced emission, Dyes Pigments 96 (2013) 495–499, https://doi.org/ [40] D.B.G. Williams, M. Lawton, Drying of organic solvents: quantitative evaluation of
10.1016/j.dyepig.2012.10.007. the efficiency of several desiccants, J. Org. Chem. 75 (2010) 8351–8354, https://
[20] F.G. Arieta, D.T. Gawne, The wettability and durability of chromium plating, Surf. doi.org/10.1021/jo101589h.
Coat. Technol. 73 (1995) 105–110, https://doi.org/10.1016/0257-8972(94)02371-9. [41] H. Chen, Y. Wu, Y. Cheng, H. Yang, F. Li, P. Yang, C. Huang, A ratiometric fluorescent
[21] S.C. Gad, G.C. Services, Environmental fate and behavior, Encyclopedia of Toxicology, sensor for zinc(II) with high selectivity, Inorg. Chem. Commun. 10 (2007)
1, 2014, pp. 600–602, https://doi.org/10.1016/B978-0-12-386454-3.00828-9. 1413–1415, https://doi.org/10.1016/j.inoche.2007.08.028.
[22] T. Agapie, Selective ethylene oligomerization: recent advances in chromium cataly- [42] D. Harvey, Modern Analytical Chemistry, McGraw-Hill Higher Education, 2000.
sis and mechanistic investigations, Coord. Chem. Rev. 255 (2011) 861–880, https:// [43] G. Liu, J. Wang, M. Yang, X. Zhang, L. Ye, F. Hao, H. Zhou, X. Tian, J. Wu, Y. Tian, Chem-
doi.org/10.1016/j.ccr.2010.11.035. ical highly selective chemosensors with versatile recognition ability caused by
[23] H. So, S. Angupillai, Y. Son, Prompt liquid-phase visual detection and low-cost minor structural change, Sens. Actuator B-Chem. 192 (2014) 586–593, https://doi.
vapor-phase detection of DCP, a chemical warfare agent mimic, Sens. Actuator B- org/10.1016/j.snb.2013.10.136.
Chem. 235 (2016) 447–456, https://doi.org/10.1016/j.snb.2016.05.106. [44] X. Cheng, Y. Yu, Y. Jia, L. Duan, Fluorescent PU films for detection and removal of Hg2
+
[24] L.M. Eubanks, T.J. Dickerson, K.D. Janda, Technological advancements for the detec- , Cr3+ and Fe3+ ions, Mater. Des. 95 (2016) 133–140, https://doi.org/10.1016/j.
tion of and protection against biological and chemical warfare agents, Chem. Soc. matdes.2016.01.103.
Rev. 36 (2007) 458–470, https://doi.org/10.1039/b615227a.