Res Chem Intermed

DOI 10.1007/s11164-015-2343-4

Photophysical properties and pH sensing applications

of luminescent salicylaldehyde derivatives

Jiaoyan Liu1 • Jinghui Cheng1 • Xiaofeng Ma1 •

Xiangge Zhou1 • Haifeng Xiang1

Received: 3 September 2015 / Accepted: 26 October 2015

 Springer Science+Business Media Dordrecht 2015

Abstract The photophysical properties, density functional theory calculations, and pH

sensing applications of a series of salicylaldehyde derivatives that contain only one small
p-conjugated benzene ring system are reported. The steric, electronic and p-extended
effects on their photophysical properties are fully investigated. The presence of a donor–
acceptor (DA) system ensures not only blue, green, and yellow emission, but also high
fluorescence quantum yields up to 0.35 in dimethylformamide (DMF) at room temper-
ature. The absorption and fluorescence properties of the salicylaldehyde derivatives are
highly dependent on the pH value of the solution, which realize their applications in pH
sensing and living cell imaging. The pKa values of these compounds ranging from 4.6 to
9.6 can be well-tuned by their structures. Moreover, in order to achieve good solubility in
water, a sodium sulfonato group was introduced into the salicylaldehyde derivatives,
which are very useful for tuning pKa values and detecting pH values in water.

Keywords Water-soluble
Substituent effect
Salicylaldehyde
Fluorescence

pH probe

Introduction

Salicylaldehyde (Sal, 2-hydroxybenzaldehyde) is a natural product in buckwheat

groats [1]. Salicylaldehyde and its derivatives are simple and commercially available
compounds, but their luminescent properties are rarely reported because they usually
contain only a very small p-conjugated benzene ring system, which would be deemed

Electronic supplementary material The online version of this article (doi:10.1007/s11164-015-2343-4)

contains supplementary material, which is available to authorized users.

& Haifeng Xiang

xianghaifeng@scu.edu.cn
1
College of Chemistry, Sichuan University, Chengdu 610041, China

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 J. Liu et al.

to be non-emissive at room temperature. In the literature, there are only limited

examples of weak luminescent salicylaldehyde derivatives [2–5] that are used as turn-
on fluorescence probes for detecting Zn2? [6], CN- [7, 8], S2- [2, 9], and amino-based
organic molecules [10, 11].
Our previous work focused on the synthesis and photophysical properties of
salicylaldehyde-based Schiff bases [12–18]. During our preparation of Schiff bases,
we noticed that salicylaldehyde derivatives exhibit weak fluorescence, as expected,
but the deprotonated (phenolic hydrogen) salicylaldehyde derivatives (Scheme 1)
have much stronger fluorescence than the correspond protonated salicylaldehyde
derivatives in solution at room temperature. To the best of our knowledge, the
photophysical properties and pH sensing applications of these salicylaldehyde
derivatives are still rarely reported, despite the fact that they are simple and common.
In the present work, we have systematically investigated the photophysical properties,
density functional theory (DFT) calculations, pH sensing applications, and living cell
imaging of a series of salicylaldehyde derivatives containing different steric hindrance
substituents (–Me and –t-butyl), electron-accepting (EA) substituents (–NO2, –F, –Cl,
–CO2H, and –SO3Na), electron-donating (ED) substituents (–Me and –NEt2), donor–
acceptor (DA) systems, and an p-extended system (naphthalene ring; Scheme 1).

Experimental section

Materials and instrumentation

All reagents were purchased from commercial suppliers and used without further
purification. UV/visible absorption spectra were recorded using a UV 765

CHO CHO CHO CHO CHO CHO CHO

6 1

5 OH OH OH OH OH OH MeO OH
2
4 3
Cl OMe Et2N MeO 5-OMe
Sal 3-Me 3-OMe 4-OMe
3-Cl 4-NEt2
CHO CHO CHO CHO
CHO CHO
OH OH HO2C OH OH
OH NaO3S OH
F NO2 5-CO2H Naph 5-S
3-F 3-NO2
3-Bu
CHO CHO CHO CHO
CHO
NaO3S OH NaO3S OH NaO3S OH NaO3S OH NaO3S
OH
Cl OMe
5-S-3-Me 5-S-3-Cl 5-S-3-OMe 5-S-3-Bu 6-S-Naph

CHO CO2H Cl NO2

−
O OH OH OH

Deprotonated Sal salicylic acid 2-chlorophenol 2-nitrophenol

Scheme 1 Chemical structures of organic molecules studied in this work

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Photophysical properties and pH sensing applications of…

Table 1 Photophysical data of the organic molecules at room temperature

Medium kabs/nm kem/ U pKa On/
(e/dm3 mol-1 cm-1) nm offb

Sal
DMF 325 (5.79 9 103); 418 (2.65 9 103) 468 0.078 8.1 98
CH3CN 255 (1.27 9 104); 325 (4.04 9 103) 402 0.0049
CH3CNa 383 (1.01 9 104) 485 0.076
3-NO2
DMF 350 (6.66 9 102); 480 (1.12 9 104) 411 0.015 6.0c –
4 3 3 d
CH3CN 244 (2.40 9 10 ); 339 (4.54 9 10 ); 460 (4.17 9 10 ) 401 \0.002
CH3CNa 449 (1.25 9 104) 403d 0.017
3-F
DMF 323 (4.80 9 103); 408 (1.04 9 103) 463 0.24 6.7 62
CH3CN 254 (1.89 9 104); 329 (4.33 9 103); 400 (7.92 9 102) 468 0.0045
CH3CNa 387 (5.62 9 103) 490 0.14
3-Cl
DMF 330 (6.92 9 103); 416 (2.47 9 103) 466 0.32 7.2 158
4 3 3
CH3CN 258 (1.12 9 10 ); 331 (3.43 9 10 ); 412 (2.00 9 10 ) 475 0.039
CH3CNa 269 (8.09 9 103); 392 (9.26 9 103) 495 0.27
3-Me
DMF 331 (7.24 9 103) 411 0.12 9.3 80
CH3CN 260 (1.59 9 104); 334 (4.38 9 103) 403 0.0071
CH3CNa 270 (1.62 9 104); 392 (1.67 9 104) 500 0.075
3-Bu
DMF 339 (3.84 9 103); 420 (6.33 9 102) 474 0.25 9.6 130
CH3CN 261 (1.17 9 104); 335 (3.31 9 103) 401 0.0073
CH3CNa 400 (4.26 9 103) 499 0.13
3-OMe
DMF 338 (3.02 9 103); 416 (2.04 9 103) 489 0.26 8.3 13
CH3CN 265 (1.74 9 104); 349 (3.85 9 103) 494 0.0067
CH3CNa 271 (9.50 9 103); 394 (9.57 9 103) 498 0.071
4-OMe
DMF 273 (1.05 9 104); 311 (4.32 9 103) 412 0.037 9.1 45
CH3CN 230 (1.38 9 104); 311 (8.60 9 103) 403 0.0085
CH3CNa 278 (1.49 9 104); 369 (1.09 9 104) 475 0.019
5-OMe
DMF 357 (3.07 9 103) 420 0.037 8.6c –
3 3
CH3CN 257 (6.98 9 10 ); 360 (2.95 9 10 ) 402 0.011
CH3CNa 415 (6.76 9 103) 540 0.017
4-NEt2
DMF 348 (1.43 9 104) 403 0.027 9.1 4.8
CH3CN 264 (9.61 9 103); 345 (5.03 9 104) 392 0.0063
CH3CNa 351 (3.99 9 104) 469 0.022

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 J. Liu et al.

Table 1 continued
Medium kabs/nm kem/ U pKa On/
(e/dm3 mol-1 cm-1) nm offb

5-CO2H
DMF 307 (2.60 9 104); 399 (5.83 9 103) 447 0.093 7.8 90
CH3CN 254 (8.56 9 103); 321 (2.19 9 103) 401 0.011
CH3CNa 249 (2.26 9 104); 279 (1.36 9 104); 382 (4.55 9 103) 494 0.090
Naph
DMF 310 (4.51 9 103); 367 (2.22 9 103) 416 (2.69 9 103); 436 405 0.021 6.4c –
(2.53 9 103)
CH3CN 317 (9.58 9 103); 360 (5.64 9 103); 415 (1.13 9 103) 403 0.0090
CH3CNa 259 (5.02 9 104); 400 (1.22 9 104) 450 0.018
5-S
DMF 333 (4.93 9 102); 416 (1.32 9 102) 478 0.13 5.9 198
CH3CN 258 (8.94 9 103); 331 (2.71 9 103) 403 0.0077
CH3CNa 261 (9.54 9 103); 385 (5.25 9 103) 495 0.090
H2 O 325 (2.44 9 103); 370 (1.13 9 103) 489 0.026
5-S-3-Cl
DMF 417 (2.54 9 103) 477 0.35 4.6 105
CH3CN 241 (8.20 9 103); 400 (2.26 9 103) 487 0.056
CH3CNa 242 (3.26 9 104); 394 (7.92 9 103) 496 0.14
H2 O 375 (3.93 9 103) 498 0.055
5-S-3-Me
DMF 340 (4.03 9 103); 416 (1.24 9 102) 487 0.13 6.4 72
CH3CN 262 (8.29 9 103); 339 (2.50 9 103) 402 0.0053
CH3CNa 241 (2.58 9 104); 388 (4.82 9 103) 503 0.076
H2 O 340 (2.18 9 103); 385 (7.42 9 102) 499 0.013
5-S-3-Bu
DMF 340 (9.52 9 102); 415 (4.07 9 102) 488 0.29 7.0 55
CH3CN 263 (5.43 9 103); 336 (1.34 9 103) 402 0.0036
CH3CNa 244 (2.48 9 104); 393 (5.31 9 103) 504 0.17
H2 O 340 (1.15 9 103) 425 0.0020
5-S-3-
OMe
DMF 412 (3.20 9 103) 493 0.25 6.3 17
CH3CN 268 (5.50 9 103); 378 (2.58 9 103) 468 0.012
CH3CNa 247 (1.46 9 104); 379 (3.58 9 103) 503 0.10
H2 O 380 (3.70 9 103) 513 0.0030
6-S-Naph
DMF 311 (4.83 9 103); 24 (4.00 9 103); 418 (3.11 9 103);437 456 0.024 6.1c –
(2.95 9 103)
CH3CN 317 (7.00 9 103); 362 (3.85 9 103) 405 0.010
a
CH3CN 271 (3.21 9 104); 401 (5.98 9 103) 448 0.021
H2 O 365 (3.39 9 103); 400 (2.44 9 103) 436 0.0020

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Photophysical properties and pH sensing applications of…

Table 1 continued
Medium kabs/nm kem/ U pKa On/
(e/dm3 mol-1 cm-1) nm offb

Salicylic acid
DMF 297 (5.99 9 103) 386 0.34 – –
CH3CN 232 (9.69 9 103); 302 (5.58 9 103) 389 0.21
CH3CNa 296 (6.70 9 103) 395 0.23
2-Chlorophenol
DMF 310 (5.60 9 10) 413 0.0048
CH3CN 275 (4.61 9 103); 281 (3.98 9 103) 407 0.0031 – –
CH3CNa 245 (1.88 9 104); 304 (6.99 9 103) 408 0.0033
2-Nitrophenol
DMF 348 (3.03 9 103); 452 (1.10 9 103) 411 0.0037 – –
CH3CN 275 (3.12 9 104); 350 (4.51 9 103) 400 \0.002
CH3CNa 276 (7.93 9 103); 425 (8.86 9 103) 392 0.0024
a
Deprotonated salicylaldehyde molecule (adding 2 equiv. of NaOH)
b
On: emission intensity at high pH value, off: emission intensity at low pH value
c
Calculated by absorption spectra due to its low emission intensity
d
Protonated and deprotonated 3-NO2 were excited at 339 nm, and the others were excited at their low-
energy absorption maxima

spectrophotometer with quartz cuvettes of 1-cm path lengths. Fluorescence spectra

were obtained using an F-7000 fluorescence spectrophotometer (Hitachi) at room
temperature. The slit width was 2.5 or 5.0 nm for both excitation and emission. The
photon multiplier voltage was 400 V. Salicylic acid, 2-chlorophenol, 2-nitrophenol
Sal, 3-NO2, 3-F, 3-OMe, 4-OMe, 4-NEt2, 5-OMe, 3-Me, 3-Bu, 3-Cl, and Naph
were purchased from J&K Chemical Company. 5-CO2H was prepared according to
the previous report [19]. All the 5-sulfosalicylaldehyde derivatives were prepared
according to our previous report [12]. Detailed synthesis information is described in
the supplementary data.

Measurement of fluorescence quantum yield (U)

U was measured by the optical dilute method with a standard of quinine sulfate
(Ur = 0.55, quinine in 0.05 mol dm-3 sulfuric acid) calculated by: Us = Ur (Br/Bs)
(ns/nr)2 (Ds/Dr), where the subscripts s and r refer to the sample and reference
standard solution, respectively; n is the refractive index of the solvents; D is the
integrated intensity. The excitation intensity, B , is calculated by: B = 1 - 10-AL,
where A is the absorbance at the excitation wavelength and L is the optical path
length (L = 1 cm in all cases). The refractive indices of the solvents at room

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 J. Liu et al.

temperature are taken from standard sources. Errors for U values (±10 %) are
estimated.

Computational details

Calculations were carried out using the Gaussian 03 software package (B3LYP
6-31G(d,p)). The geometry optimization, absorption transition and spectrum were
carried out by DFT and TD-DFT, respectively. The theoretical modelling was
performed in the isolated molecule approximation ignoring the effect of the
aggregation state or solvent. For the atoms of organic materials, the standard split-
valence basis sets 6-31G(d,p) augmented with polarization d-functions for the non-
hydrogen atoms and p-functions for the hydrogen atoms were used. Full geometry
optimization of all the organic materials corresponding to the minima on the
potential energy surface (PES) was conducted until a gradient of 10-5 at.u. The spin
multiplicities of all the salicylaldehyde derivatives were set equal to 1. The charges
of the protonated and deprotonated salicylaldehyde derivatives without sulfonato
group were set equal to 0 and -1, respectively, and those of the protonated and
deprotonated 5-sulfosalicylaldehyde derivatives (–SO- 3 ) were set equal to -1 and
-2, respectively. The other parameters were set to default values.

Measurement of pH sensing

The pH responses of the salicylaldehyde derivatives (1.0 9 10-4 or 5.0 9

10-5 mol dm-3) without and with a sodium sulfonato group were measured in
the mixed solvents of aqueous B–R buffer solution (a mixture of 0.04 mol/L
H3BO3, H3PO4 and CH3COOH in water)/DMSO (9:1 by volume) and pure aqueous
B–R buffer solution, respectively. The interference experiments were performed
under similar conditions: the concentrations of 5-S-3-Cl were all kept at 1.0 9 10-4
mol dm-3 and two equivalents of metal ions were added. All types of absorption

Fig. 1 Absorption spectra of 3 (a)

6.0x10 0.14
Sal and deprotonated Sal
(adding 2 equiv. of NaOH) in
3
MeCN (a calculation, 4.0x10 0.09
b experiment)
f
ε / dm3 mol−1cm−1

3
2.0x10 0.05

0.0 0.00
(b)
1.5x10
4 Sal
deprotonated Sal
4
1.0x10
3
5.0x10

0.0
200 250 300 350 400 450 500 550
Wavelength / nm

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Photophysical properties and pH sensing applications of…

Fig. 2 Energy level diagrams

of Sal (a) and deprotonated Sal
(b) with their frontier molecular
orbitals

and fluorescence measurement were monitored at least 1 h after the addition of the
probe or probe/metal ion to the buffer solution at room temperature.

Cell culture methods and imaging

The imaging of HeLa cells was finished by a fluorescence vertical microscope

(LEICA DM2500). HeLa cells were cultured in Dulbecco’s modified eagle medium
(DMEM) supplemented with 10 % fetal bovine serum, penicillin (100 units mL-1),
streptomycin (100 mg mL-1) and 5 % CO2 at 37 C. After removing the incubating
media and rinse with PBS for three times, the cells were incubated with the dye
(1.0 9 10-5 mol dm-3) in PBS for 2 h at room temperature. Then, the cells were
washed three times with PBS and incubated with aqueous alkali for 20 min. Finally,
the cells were imaged with a confocal microscope.

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 J. Liu et al.

Fig. 3 Normalized absorption 0.3

(a) Sal 3-F
spectra of 3-substituted 3-NO2 3-Bu
protonated (a) and deprotonated 0.2
(b) salicylaldehyde derivatives 3-Cl
in MeCN
0.1

Absorbance
0.0
(b) 3-Me
0.2 3-OMe

0.1

0.0
300 350 400 450 500 550
Wavelength / nm

Results and discussion

Synthesis and characterization

Most of the salicylaldehyde derivatives without a sodium sulfonato group in the

present work are commercially available. For the purpose of comparison, salicylic
acid, 2-chlorophenol, and 2-nitrophenol (Scheme 1) were examined as well. For
some cases, especially for sensing applications, the preferred work conditions are
aqueous media, but these salicylaldehyde derivatives have poor solubility in water.
Furthermore, our previous work [12] revealed that the presence of sodium sulfonato
groups in Salen Schiff bases ensures not only good stability but also solubility in
water without affecting the excited state properties. Therefore, 5-sulfosalicylalde-
hyde derivatives were prepared according to our previous method [12]. Detailed

Fig. 4 Normalized absorption 0.3

(a)
spectra of other substituted
protonated (a) and deprotonated 4-OMe
(b) salicylaldehyde derivatives
0.2
5-OMe
in MeCN 4-NEt2
0.1
Absorbance

0.0
(b)
0.2
Naph
5-CO2 H
0.1

0.0
300 350 400 450 500 550
Wavelength / nm

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Photophysical properties and pH sensing applications of…

synthesis information is described in the supplementary data. All of these

sulfosalicylaldehyde derivatives have good solubilities in water, as expected. For
example, 1 mL water can dissolve at least 100 mg of 5-S-3-Cl. The deprotonated
salicylaldehyde derivatives are feasibly obtained by adding NaOH, because the
protonated salicylaldehyde derivatives are weak acids. This deprotonation process
can be confirmed by proton nuclear magnetic resonance (1H NMR) analysis. The 1H
NMR signals of 3-Me (d = 11.32 ppm in CD3CN) belonging to the –OH
disappeared after adding a base (Fig. S1 in the supplementary data). This
deprotonation process is also confirmed by the fact that the resultant quinine-type
phenolate anion (phenolic O-) has lower energy absorption and emission
enhancement (see subsequent discussion). Most of the salicylaldehyde derivatives
in this work are stable either in solution or in a solid state under air during synthesis,
characterization, and application.

Electronic absorption spectroscopy

The ultraviolet (UV)/visible absorption data of all the salicylaldehyde derivatives

are listed in Table 1. The absorption spectra of Sal and deprotonated Sal in MeCN

Fig. 5 Normalized absorption 0.3

spectra of protonated (a) and
(a)
5-S-3-Cl
deprotonated (b) 5- 5-S-3-Me
0.2
sulfosalicylaldehyde derivatives
in MeCN
5-S
0.1
Absorbance

0.0
(b) 5-S-3-OMe
0.2 5-S-3-Bu
6-S-Naph
0.1

0.0
300 350 400 450 500 550
Wavelength / nm

Fig. 6 Photographs (top under sunlight, bottom under 360-nm UV light) of the selected salicylaldehyde
derivatives (1.0 9 10-4 mol dm-3 in DMF)

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 J. Liu et al.

are given in Fig. 1. Sal shows two absorption maxima (kabs) at 255 and 325 nm with
a molar extinction coefficient (e) of 1.26 9 104 and 4.04 9 103 dm3 mol-1 cm-1,
respectively. The absorption spectrum is reproduced well using gas-phase, time-
dependent-DFT (TD-DFT) calculations which predict two absorption maxima at
239 and 324 nm with an oscillator strength (f) of 0.1535 and 0.0604, respectively
(Fig. 1a). The lower energy absorption (S0 ? S1) mainly corresponds to the highest
occupied molecular orbital (HOMO) ? lowest unoccupied molecular orbital
[LUMO; wavefunction coefficient (WFC) = 0.59] of salicylaldehyde, and the
higher energy absorption (S0 ? S2) mainly arises from a transition between
HOMO-1 and LUMO (WFC = 0.65). The energy level diagram of Sal with orbital
isosurfaces is given in Fig. 2. Based on these frontier molecular orbitals of Sal, both
lower and higher energy absorptions can mainly be assigned to p ? p* transitions
with a small contribution from n ? p* of a phenolic oxygen atom for the higher
energy absorption. Moreover, a weak intramolecular charge transfer (ICT) from the
donor of –OH to the acceptor of –CHO is also observed in the transition between
HOMO and LUMO (Fig. 2a). On the other hand, compared with Sal, deprotonated
Sal (Fig. 1b) exhibits a stronger red-shifted absorption band (kabs = 383 nm,
e = 8.66 9 103 dm3 mol-1 cm-1), which is well-matched with TD-DFT calcula-
tions that simulate one absorption maxima at 390 nm with an f of 0.0876. The
phenomenon of a stronger red-shifted spectrum can be reasonably explained by the
fact that electron-rich phenolate anions have resonance structures of quinine
(Fig. S2). These resonance structures lead the p-function frontier molecular orbitals
to a uniform distribution in the whole molecule skeleton of deprotonated Sal, and
consequently deprotonated Sal has not only a smaller energy gap between the
HOMO and LUMO but also an increment of molar extinction co-efficient. This also
is the reason why the salicylaldehyde derivatives are acid. It is noteworthy that the
absorption (S1 ? S0) of deprotonated Sal does not mainly arises from a transition
of HOMO ? LUMO, but HOMO-1 ? LUMO (WFC = 0.62). Obviously, an
intense DA charge transfer from the electron-rich phenolate anion fragment to the
electron-deficient benzene ring center is found in this transition. As depicted in

Fig. 7 Normalized emission (a) Sal

spectra of 3-substituted
protonated (a) and deprotonated 3-NO2
Emission intensity / a.u.

(b) salicylaldehyde derivatives

in MeCN. Protonated and
3-Cl
deprotonated 3-NO2 was not 3-Me
excited at 460 or 449 nm, but at
339 nm, because no emission
was observed if excited at 460 or (b) 3-OMe
449 nm, and the others were 3-F
excited at their low-energy
absorption maxima
3-Bu

400 450 500 550 600 650

Wavelength / nm

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Photophysical properties and pH sensing applications of…

Fig. 8 Normalized emission (a) 4-OMe

spectra of other substituted
protonated (a) and deprotonated 5-OMe
4-NEt2

Emission intensity / a.u.

(b) salicylaldehyde derivatives
in MeCN (excited at their low-
energy absorption maxima) 5-CO2 H

(b) Naph

400 450 500 550 600 650

Wavelength / nm

Fig. 9 Normalized emission

spectra of protonated (a) and
(a) 5-S-3-OMe
deprotonated (b) 5- 5-S-3-Bu
Emission intensity / a.u.

sulfosalicylaldehyde derivatives
in MeCN (excited at their low-
6-S-Naph
energy absorption maxima)

(b) 5-S
5-S-3-Cl
5-S-3-Me

400 450 500 550 600 650 700

Wavelength / nm

Fig. 2b, in the frontier molecular orbitals of deprotonated Sal, the phenolate anion
donor is mostly contributed to HOMO-1, whereas the benzene ring acceptor is
mostly contributed to LUMO, clearly indicating the intense DA charge transfer from
the donor end groups to the central acceptor fragment. This DA charge transfer is
useful for red shifting absorption and emission bands [14, 20, 21].
The absorption spectra of protonated and deprotonated Sal, 3-OMe, 3-NO2, 3-F, 3-
Cl, 3-Me, and 3-Bu in MeCN are shown in Fig. 3 to investigate the 3-substituted
effects on electronic absorption properties. Compared with Sal, the presence of methyl
and t-butyl groups has little effect on the absorption spectrum. The presence of a strong
ED –OMe, or weak EA –Cl, or –F (see subsequent discussion) substitute brings about
small red shifts in the absorption spectra. It is obvious that, in the region more than
300 nm, 3-NO2 containing a strong EA substitute has two absorption peaks with the
lowest-energy absorption band (kabs = 460 nm). When deprotonated, except 3-NO2,

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 J. Liu et al.

these salicylaldehyde derivatives give red shifts in the absorption as in the above
discussion.
4-Substituted, 5-substituted, and p-extended effects on the absorption spectra of
protonated and deprotonated salicylaldehyde derivatives were examined as well, as
shown in Fig. 4. For protonated salicylaldehyde derivatives, 4-substituted -OMe
(4-OMe, kabs = 311 nm) and 5-substituted –CO2H (5-CO2H, kabs = 321 nm)
exhibits blue shifts in the absorption spectrum, compared with Sal (kabs = 325 nm).
On the contrary, 3-substituted (3-OMe, kabs = 349 nm), 5-substituted –OMe (5-
OMe, kabs = 360 nm), and 4-substituted –NEt2 (4-NEt2, kabs = 345 nm) show red
shifts. As expected, the p-extended system in Naph (kabs = 360 nm) displays a red-
shifted absorption spectrum. When deprotonated, these salicylaldehyde derivatives
give red shifts in the absorption similar to the 3-substituted salicylaldehyde
derivatives.
In order to improve the solubility in water, a sodium sulfonato group was
introduced to the 5-position of salicylaldehyde derivatives. Unlike the results of our
previous work showing that sodium sulfonato groups in Salen Schiff bases had little
effect on the excited state properties [12], the presence of a 5-substituted sodium
sulfonato group in salicylaldehyde derivatives has some obvious effects on the
absorption spectra. As shown in Fig. 5, since a sulfonato group is a strong EA
substituent (see subsequent discussion), most 5-sulfosalicylaldehyde derivatives
have red-shifted absorption spectra compared with the corresponding salicylalde-
hyde derivatives. After deprotonation, most 5-sulfosalicylaldehyde derivatives also
exhibit red shifts in the absorption spectra as expected.

Fluorescence spectroscopy

The fluorescence emission data of the salicylaldehyde derivatives in solution are

listed in Table 1. At room temperature, the protonated and deprotonated
salicylaldehyde derivatives in the present work emit blue, green, and yellow light
upon UV excitation, as shown in Fig. 6 and Table 1.
As examples, the normalized fluorescence emission spectra of 3-substituted
salicylaldehyde derivatives including Sal, 3-NO2, 3-Cl, 3-Me, 3-OMe, 3-Bu, and
3-F in MeCN are given in Fig. 7. These salicylaldehyde derivatives emit broad
fluorescence bands in the region from 375 to 600 nm, mostly attributed to p ? p*
transition similar to their absorption transition. A tendency of kem is observed in the
order of Sal & 3-Bu & 3-Me \ 3-F \ 3-Cl \ 3-OMe, which is consistent with
the nature of steric hindrance substituents of –Me and –Bu, and EA or ED
substituents of –F, –Cl, and –OMe [22]. After deprotonation, these 3-substituted
salicylaldehyde derivatives also give red-shifted emission, as expected, and Sal,
3-Me, and 3-Bu exhibit distinct red shifts (Dkem & 90 nm). It is a special case that
both protonated and deprotonated 3-NO2 have blue-shifted emission spectra, even
though they have the most red-shifted absorption spectra. It should be noted that for
fluorescence emission measurement, protonated (kem = 401 nm) and deprotonated
(kem = 403 nm) 3-NO2 were excited at 339 nm but not their low-energy absorption
maxima (kabs = 460 and 449 nm protonated and deprotonated Sal, respectively); in
contrast, the other compounds were excited at their low-energy absorption maxima

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Photophysical properties and pH sensing applications of…

Fig. 10 Optimized structures and frontier molecular orbitals for the selected protonated (a) and
deprotonated (b) salicylaldehyde derivatives calculated at a B3LYP 6-31G(d,p) level of theory

(Table 1). If protonated and deprotonated 3-NO2 were excited at their low-energy
absorption maxima, the resultant emission intensity was too weak to measure.
4-Substituted, 5-substituted, and p-extended effects on the emission spectra of
protonated and deprotonated salicylaldehyde derivatives were examined as well, as
shown in Fig. 8. For protonated salicylaldehyde derivatives, 4-OMe, 5-OMe,
4-NEt2, 5-CO2H, and Naph exhibit the similar emission spectra as Sal
(kem = 401–405 nm) with an exception of the shoulder emission band (kem =
518 nm) for 4-NEt2. When deprotonated, these salicylaldehyde derivatives emit
low-energy, as expected. It is observable that the emission band of deprotonated
5-OMe red shifts into the yellow region (kem = 540 nm). However, the emission
band of protonated and deprotonated Naph containing the biggest p-extended
system is 403 and 450 nm, respectively, which is beyond our expectation.
As shown in Fig. 9, most 5-sulfosalicylaldehyde derivatives have red-shifted
emission spectra compared with the corresponding salicylaldehyde derivatives
without a sulfonato group (Fig. 7), which is consistent with the tendency in their
absorption spectra. After deprotonation, these 5-sulfosalicylaldehyde derivatives
also exhibit red shifts in the emission spectra as expected.
All fluorescence quantum yields of these salicylaldehyde derivatives in solution
were measured by the optical dilute method of Demas and Crosby [23] with a
standard of quinine sulfate (Ur = 0.55, quinine in 0.05 mol dm-3 sulfuric acid) and
are listed in Table 1. The emission intensity of Sal in MeCN is very weak, and, thus,
its U in MeCN is 0.0049. However, if DMF solvent is adopted, the U of Sal would
be enhanced up to 0.078, because DMF is more basic than MeCN, resulting in the
formation of deprotonated Sal. The pH value of the used DMF is about *8. DMF
might not be basic enough to deprotonate all the compounds (pKa = 4.6–9.6, see
below), but the alkalinity of DMF would help improve emission intensity.
Moreover, the solvent effect would affect emission intensity and it should be
considered. If NaOH is added, a similar enhancement can be also observed in

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MeCN (Table 1), which confirms the deprotonation-induced fluorescence enhance-

ment. As depicted in Fig. S9, the emission intensity would be increased about
30-fold upon adding OH- to the MeCN solution of Sal, and then the enhanced
emission would be quenched efficiently (1/30) if H? is added. Moreover, the above
reactions of deprotonation and protonation are rapid and reversible. The phenomena
of deprotonation-induced fluorescence enhancement can be observed in all the
salicylaldehyde derivatives (Table 1).
3-Position hindrance substituents (–Me and –t-butyl) and weak EA (–Cl, –F) or
strong ED substituents (–Me) would be in favor of enhancing the U. The U of Sal,
3-Me, 3-Bu, 3-Cl, 3-F, and 3-OMe in DMF is 0.078, 0.12, 0.25, 0.32, 0.24, and
0.26, respectively. On the contrary, a 3-position strong EA substituent (–NO2)
would decrease the U. The U of 3-NO2 in DMF is 0.015. 4-Position ED substituents
would not improve the U (0.027 and 0.037 for 4-NEt2 and 4-OMe, respectively). A
5-position ED substituent (–Me) and EA substituent (–CO2H) also have opposite
effects on the U. The U values of 5-OMe and 5-CO2H in DMF are 0.037 and 0.093,
respectively. Unexpectedly, Naph containing the biggest p-extended has a low U of
0.021. For 5-sulfosalicylaldehyde derivatives, we found that the presence of a
sodium sulfonato group has little effect on the U. 5-S-3-Cl has the highest U, up to
0.35, in DMF.

Electronic structure calculations and proposed mechanism

To gain further insight into the nature of the excited states, DFT calculations were
carried out for all the salicylaldehyde derivatives with the Gaussian 03 program
package [B3LYP 6-31G(d,p)] [24]. The geometry optimization, absorption transi-
tion and spectrum were carried out by DFT and TD-DFT. The spin multiplicities of
all the salicylaldehyde derivatives were set equal to 1. The charges of the protonated
and deprotonated salicylaldehyde derivatives without a sulfonato group were set
equal to 0 and -1, respectively, and those of the protonated and deprotonated
5-sulfosalicylaldehyde derivatives (–SO- 3 ) were set equal to -1 and -2, respec-
tively. The theoretical modelling was performed in the isolated molecule
approximation ignoring the effect of the aggregation state or solvent.
The optimized structures of protonated and deprotonated Sal, 3-NO2, 3-Cl,
3-OMe, 3-Me, Naph, and 5-S are shown in Fig. 10 and those of the others are
depicted in Fig. S3. The optimized geometries of Sal and 3-OMe remain similar to
their single crystal structures (Fig. S10). In the optimized geometry, the
intramolecular hydrogen bonds between H atoms in –OH and O atoms in –CHO
are found in both Sal and 3-OMe, as expected.
Diagrams of the LUMO ? 1, LUMO, HOMO, and HOMO-1 for the ground
states and the energies of frontier molecular orbitals of the selected salicylaldehyde
derivatives are shown in Fig. 10 and S3 and listed in Table S1 and S2, respectively.
The LUMO ? 1, LUMO, HOMO, and HOMO-1 spatial plots of 3-Me and 3-Bu are
similar to these of Sal except that 3-Me and 3-Bu have a very small contribution to
p-functions of HOMO, and HOMO-1, revealing that methyl and t-butyl are steric
hindrance substituents with very weak ED properties. Sal, 3-Me, and 3-Bu have
similar levels of LUMO ? 1, LUMO, HOMO, and HOMO-1 (Table S1 and

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Photophysical properties and pH sensing applications of…

Fig. S4). Consequently, they have similar energy gaps and absorption and emission
spectra (Figs. 3, 7). In the frontier molecular orbitals of 3-OMe, 4-NEt2, and
3-NO2, the strong ED –OMe and –NEt2 substituents are mostly contributed to
HOMO and HOMO-1 orbitals, whereas strong EA –NO2 substituent is mostly
contributed to LUMO and LUMO ? 1 orbitals, clearly indicating their nature of ED
and EA properties, respectively. In the frontier molecular orbitals of 3-F and 3-Cl,
the weak ED –F and –Cl substituents have similar contributions to HOMO/HOMO-
1 and LUMO/LUMO ? 1 orbitals. In some cases, –F and –Cl are weak EA
substituents, because their electronegativities are bigger than that of –H. In some
other case, nonetheless, –F and –Cl substituents can donate their lone pair electrons
through p–p conjugation, and, thus, they might have ED properties. Although these
ED and EA substituents lead their absorption and emission spectra to red shifts
(except that 3-NO2 is non-emissive when excited at the low-energy absorption
maximum of 460 nm), the causes are different. The EA –F and –Cl substituents
have little change in the level of HOMO and the lower level of LUMO, but the ED –
OMe substituent has an opposite effect. The ED –OMe substituent has little effect
on the level of LUMO and the higher level of HOMO. The EA –NO2 substituent
leads not only to some lower levels of HOMO but also to the much lower level of
LUMO. After deprotonation, however, an intense DA charge transfer (HOMO-
1 ? LUMO) from the electron-rich phenolic O- fragment to the electron-deficient
center of the benzene ring is found in these salicylaldehyde derivatives, as
previously mentioned, resulting in red-shifted absorption and emission spectra.
The 4-substituted, 5-substituted, and p-extended effects on the HOMO and
LUMO levels of protonated and deprotonated salicylaldehyde derivatives are
examined as well (Figs. S5, S6). For example, the presence of an ED –OMe
substituent at different positions has different effects on the levels HOMO and
LUMO. Compared with Sal, 4-OMe has similar levels of HOMO and LUMO,
resulting in similar absorption and emission spectra. 3-OMe and 5-OMe have little
effect on the level of LUMO and the higher level of HOMO, resulting in the red-
shifted absorption and emission spectra.
Our previous work [12] revealed that the photophysical properties of the
sulfonato-Salen Schiff bases are similar to those of the Salen ligands without
sulfonato groups (SO- 3 ), because the contribution of sulfonato groups to the
LUMO ? 1, LUMO, HOMO, and HOMO-1 is relatively small. For these
5-sulfosalicylaldehyde derivatives containing much simpler molecular structures,
however, a sulfonato group induces dramatic effects on the LUMO ? 1, LUMO,
HOMO, and HOMO-1 spatial plots (Figs. 10 and S3). The sulfonato group strongly
attracts an electron cloud of the benzene ring, resulting in there being almost are no
p-functions in the benzene ring. The calculated LUMO ? 1, LUMO, HOMO, and
HOMO-1 levels of all the 5-sulfosalicylaldehyde derivatives are much higher than
those of neutral salicylaldehyde derivatives without a sulfonato group. Especially,
their LUMO ? 1 and LUMO levels are positive, which might be caused by the
effect of a -1 charge in the 5-sulfosalicylaldehyde derivatives [12]. A similar
phenomenon can be observed in deprotonated salicylaldehyde derivatives. If the
sulfonato group was revised from –SO- 3 to –SO3H, the charge would be equal to 0,
which would help the levels of LUMO ? 1, LUMO, HOMO, and HOMO-1 to

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 J. Liu et al.

(a) (b)
0.6 4.2
A380/A328
0.4
10 0.5 3.5
A380
pH

Absorbance
0.4 2.8
Absorbance

0.3

A380/A328
1 0.3 2.1
0.2 A328
0.2 1.4

0.1 0.1 0.7

0.0 0.0
0.0
300 350 400 450 500 1 2 3 4 5 6 7 8 9
Wavelength / nm pH
(c) (d)
10

Emission intensity / a.u.

Emission intensity / a.u.

pH

400 450 500 550 600 650 1 2 3 4 5 6 7 8 9 10

Wavelength / nm pH

Fig. 11 Absorption (a) and emission (c) spectra (excited at 380 nm) and plots of absorption (b; A325 and
A378 are the absorbance at wavelengths of 328 and 380 nm, respectively) and emission intensity
(d) (kem = 500 nm) of 5-S-3-Cl (1.0 9 10-4) versus pH value in the pure aqueous B–R buffer solution

change into normal values (Fig. S7). The calculated energy gaps of 5-sulfosalicy-
laldehyde derivatives are smaller than these of the salicylaldehyde derivatives
without a sulfonato group, which is consistent with the tendency observed in their
absorption and emission spectra.
The fluorescence quantum yields of protonated salicylaldehyde derivatives are
very low. The weak fluorescence was attributed to the free rotation of the carbonyl
group in salicylaldehyde derivatives which induces fast fluorescence decay in the
previous work [6]. However, the X-ray single crystal structures (Fig. S10) and
theoretical optimized structures (Fig. 10) have demonstrated that the intramolecular
hydrogen bond between an H atom in –OH and an O atom in –CHO exists and
prevents the free rotation of the carbonyl group. As previously discussed, a weak
ICT from the electron-rich –OH to the electron-poor –CHO is observed (Fig. 2a).
And, thus, we firstly assigned that this ICT might be responsible for the weak
fluorescence for salicylaldehyde derivatives, but when we carefully compared the
frontier molecular orbitals of Sal and salicylic acid, we found that it is not so. As
shown in Fig. S8, Sal and salicylic acid have the similar frontier molecular orbitals
and transitions. Moreover, such an ICT from the electron-rich –OH to the electron-
poor –CO2H is also observed in salicylic acid, which does not match the fact that

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Photophysical properties and pH sensing applications of…

salicylic acid has a high U of 0.21 in MeCN. The calculated lower energy absorption
(S0 ? S1, HOMO ? LUMO) of salicylic acid occurs at 304 nm (302 nm in
MeCN) and has a bigger f (0.0745) than that of Sal (0.0604). These different values
in f might be one of the reasonable factors for low and high U for Sal and salicylic
acid, respectively. After deprotonation, an intense DA charge transfer from the
electron-rich phenolic O- fragment to the electron-deficient center of a benzene ring
is found in the HOMO-1 ? LUMO transition (S1 ? S0, f = 0.0876) of deproto-
nated Sal. Therefore, we tentatively assign the strong fluorescence of deprotonated
Sal to this DA system. Furthermore, protonated and deprotonated 2-chlorophenol
and 2-nitrophenol have low fluorescence quantum yields (Table 1), indicating that
–CHO and –CO2H substituents play important roles in enhancing the fluorescence
of phenol. It should be noted that deprotonation would eliminate the excited state
intramolecular proton transfer (ESIPT) process from –OH to –CHO, which might
lead to fluorescence enhancement as well. All the above calculations and
experimental results provide unequivocal insights into the inherent relationship
between photophysical properties and chemical structures, and useful tools for
tuning absorption and emission bands.

Optical pH probes

Protons are one of the most important targets among the intracellular species of
interest, as it is well known that pH plays a central role in many cellular events. In
general, there are two main kinds of pH probes. One for cytosol works at a pH of
about 6.8–7.4, and the other for acidic organelles, such as lysosomes and
endosomes, functions over the pH range of about 4.5–6.0 [15, 25–35]. However,
most optical pH probes, especially for fluorescent pH probes, suffer from several
drawbacks. For example, optical probes allow precise measurement of pH in a much
narrower region. It is difficult to predict and tune pKa by modifying chemical
structures. Probes have a bad solubility in pure water and, thus, organic solvents or
mixed solvents of water and organic solvents are needed. Therefore, designing
optical pH probes that are simple, water-soluble, and good at tuning pKa is still of
great importance.
The absorbance and fluorescence intensity of the salicylaldehyde derivatives
highly depend on the pH values of the solution and, thus, they can be used as optical
pH probes. The pH responses of the salicylaldehyde derivatives without and with
sodium sulfonato groups were measured in the mixed solvents of aqueous Britton–
Robinson (B–R) buffer solution (a mixture of 0.04 mol/L H3BO3, H3PO4 and
CH3COOH in water)/dimethylsulfoxide (DMSO, 9:1 by volume) and pure aqueous
B–R buffer solution, respectively. As examples, the pH responses of 5-S-3-Cl in the
pure aqueous B–R buffer solution are demonstrated in Fig. 11 to describe the pH
sensing behaviors of these salicylaldehyde derivatives. The absorption and emission
properties of 5-S-3-Cl are strongly dependent on pH values. The absorbance at
328 nm (A328) that belongs to the protonated 5-S-3-Cl remains constant at
pH \ 3.6. And then it reduces along with the increase of pH values from 3.6 to 5.7
and finally remains constant again at pH [ 5.7 (Fig. 11a). On the contrary, A380
belonging to the deprotonated 5-S-3-Cl has totally adverse pH responses, which are

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 J. Liu et al.

Fig. 12 Brightfield (a; 9200) and fluorescence images (b) of 3-Cl-stained HeLa cells and fluorescence
images of cells upon adding OH- (c) and H? (d; excited at 420 nm)

zero, increased, and constant at pH \2.7, 2.7–6.6, [6.6, respectively. Both plots of
A328 and A380 versus pH value can be used to detect pH values through colorimetric
measurement (Fig. 11b). Moreover, the ration between A380 and A328 exhibits
similar pH responses with A380. Generally, this ratiometric method of detection is
more desirable due to its advantages of selectivity, insensitivity to ambient or
scattered light, and elimination of instrumental fluctuation [36]. The above facts
reveal that the pKa of 5-S-3-Cl is about 4.6. Like its absorption spectra, the emission
spectra of 5-S-3-Cl have two emission peaks at different pH values, in which one is
kem = 433 nm originating from protonated 5-S-3-Cl at low pH values, the other is
kem = 500 nm coming from deprotonated 5-S-3-Cl at high pH values. Since the
emission intensity of protonated 5-S-3-Cl is much less than that of deprotonated 5-
S-3-Cl, only one emission peak (kem = 500 nm) is apparent in Fig. 11c. As shown
in Fig. 11d, the fluorescence is turn-off, increased, and turn-on (saturated) at pH
\2.7, 2.7–6.5,[6.5, respectively, revealing that the pKa of 5-S-3-Cl also is 4.6. The
emission intensity ratio between turn-on and turn-off fluorescence is 105 and the U
of deprotonated 5-S-3-Cl (pH [ 6.5) is 0.24, indicating its potential applications in
pH sensing and cell imaging for cytosol and acidic organelles.
The pH responses of the other salicylaldehyde derivatives are depicted in
Figs. S11–S27. The pKa values (4.6–9.6) of all the salicylaldehyde derivatives are
summarized in Table 1, which were calculated by the fluorescence method, except
that 5-OMe, 3-NO2, Naph, and 6-S-Naph have low fluorescence and their pKa
values were calculated by the absorption method. It is well-known that phenol is a
weak acid with a pKa of about 10 [37]. Sal (pKa = 8.1) with the presence of an EA –
CHO substituent at the ortho position is more acidic than phenol, because the
inductive effect of the EA substituent reduces the electron density of the benzene

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Photophysical properties and pH sensing applications of…

Fig. 13 Brightfield and fluorescence images (9600) of HeLa cells (pH * 7.0) stained with 3-Cl
(excited at 420 nm) and a lysosomal dye (LysoTracker) or a mitochondrial dye (MitoTracker; excited at
540 nm)

ring, resulting in the negative charge of phenolic hydroxyl delocalizing into the
benzene ring. Consequently, the resultant phenolate anion is more stable and readily
releases protons. On the other hand, ED substituents stress adverse effects to
decrease the acidity.
As listed in Table 1, for the 3-substituted salicylaldehyde derivatives, a tendency
of pKa is observed in the order of 3-NO2 (pKa = 6.0) \ 3-F (pKa = 6.6) \ 3-Cl
(pKa = 7.3) \ Sal (pKa = 8.1) \ 3-OMe (pKa = 8.3) \ 3-Me (pKa = 9.3) \
3-Bu (pKa = 9.6), which is consistent with the fact that EA and ED substituents
cause the acidity to increase and reduce, respectively, as previously mentioned.
Among these salicylaldehyde derivatives, 3-Me and 3-Bu are the weakest acids.
Methyl and t-butyl are not only weak ED substituents but also steric hindrance
substituents. These ortho-position steric hindrance substituents will prevent
phenolic hydroxyl from dissociating through solvation. As expected, 4-OMe
(pKa = 9.1), 4-NEt2 (pKa = 9.1), and 5-OMe (pKa = 8.6) containing ED sub-
stituents have a weaker acidity than Sal, but 5-CO2H (pKa = 7.8) containing a
weak EA substituent has a stronger acidity than Sal. However, like its emission
property, Naph has a pKa of 6.4, which also is beyond our expectation. Remarkably,
the presence of a strong EA 5-substituted sulfonato group increases the acidity
greatly. These 5-sulfosalicylaldehyde derivatives have much stronger acidity

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 J. Liu et al.

(pKa B 7.0) in the order of 5-S-3-Cl (pKa = 4.6) \ 5-S-3-OMe (pKa = 5.4) \ 5-S
(pKa = 5.9) \ 6-S-Naph (pKa = 6.1) \ 5-S-3-OMe (pKa = 6.3) \ 5-S-3-Me
(pKa = 6.4) \ 5-S-Bu (pKa = 7.0), which is roughly consistent with the above
observed rules.
On account of the complexity of intracellular and other environments, an
additional examination of the probe was performed to determine whether metal ions
potentially interfered. It is well known that phenolic hydroxyl can bind with many
metal ions in solution. In order to evaluate the selectivity of 5-S-3-Cl (Fig. S28), 14
different kinds of metal ions (Ag?, Al3?, Cd2?, Ce3?, Co2?, Cr3?, Cu2?, Fe3?, Li?,
Mn2?, Ni2?, Pb2?, Sr2?, Zn2?), which probably interfere with pH detection, were
performed under the similar conditions: the concentrations of 5-S-3-Cl were all kept
at 1.0 9 10-4 mol dm-3 and two equivalents of metal ions were added. Both at
pH = 4.6 and 7.0, among these metal ions, only Cu2? had an obvious emission
quenching effect. The combined results reveal that these salicylaldehyde derivatives
have high emission intensity ratios (up to about 200) between turn-on and turn-off
fluorescence, small interference from metal ions, and well-tuned pKa values
(4.6–9.6), and, thus, they have useful and potential applications in pH sensing and
cell imaging for both cytosol and acidic organelles.

Living cell imaging

Fluorescence pH probes that can selectively monitor specific pH value in living cells
have been indispensable tools for understanding biological phenomena. In order to
evaluate the possibility of 3-Cl as a fluorescence pH probe to image living cells,
3-Cl was firstly used to culture HeLa cells in phosphate buffered saline (PBS) for
20 min. Through a fluorescence microscope, the untreated A549 cells showed no
fluorescence, as shown in Fig. 12a. After sole incubation of HeLa cells with 3-Cl,
weak green intracellular fluorescence (Fig. 12b, excited at 420 nm) was observed,
because HeLa cells are cervical cancer cells that are more acid (pH = *5) than
normal cells and the pKa value of 3-Cl is 7.2. If NaOH was added (pH = *11),
3-Cl would be deprotonated to turn on the fluorescence (deprotonated 3-Cl emits
strong green fluorescence with kem = 505 nm in aqueous B-R buffer solution/
DMSO, Fig. S18), and then the bright and green cell image was captured (Fig. 12c).
However, if HCl was added (pH = *3), the fluorescence would be quenched
efficiently (Fig. 12d). We also found that 3-Cl has little cytotoxicity to HeLa cells,
because the 3-Cl-stained HeLa cell is still alive and cannot be further stained by
trypan blue. The combined results reveal that 3-Cl is suitable for a fluorescence pH
probe and living cell imaging.
In order to reveal the location of the probe after it enters living cells, co-staining
studies of 3-Cl with a lysosomal dye (LysoTracker) and a mitochondrial dye
(MitoTracker) were conducted (Fig. 13). The typical bright field image (Fig. 13a),
luminescent image with 3-Cl (Fig. 13b) and LysoTracker (Fig. 13c), and the
corresponding merged image (Fig. 13d) indicate that 3-Cl is weakly overlapped
with LysoTracker. On the other hand, the luminescent overlay image (Fig. 13h) of
3-Cl (Fig. 13f) and MitoTracker (Fig. 13g) shows that some yellow regions are
present, indicating that 3-Cl and MitoTracker stain the same organelles. These

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Photophysical properties and pH sensing applications of…

results suggest that that 3-Cl is located in mitochondria and lysosomes in addition to
the cytosol (Fig. 12c).

Conclusions

We have systematically synthesized and studied the photophysical, pH sensing, and

living cell imaging properties of a series of salicylaldehyde derivatives. Although
these salicylaldehyde derivatives contain a very small p-conjugated benzene ring,
they have many advantages, such as facile preparation and modification of chemical
structures, tunable fluorescence, high fluorescence quantum yields (up to 0.35),
emission intensity ratios (up to about 200) between turn-on and turn-off
fluorescence, small interference from metal ions, and well-tuned pKa values
(4.6–9.6). Therefore, we believe that these novel kinds of simple salicylaldehyde
derivatives provide a new paradigm in the design of optical materials and have
useful and potential applications. Further studies on salicylic acid molecules and the
extension of fluorescence bands of these salicylaldehyde derivatives into the red or
near-infrared regions are currently underway in our laboratory.

Acknowledgments This work was supported by the National Natural Science Foundation of China
(nos. 21172160 and 21372169). We thank the College of Life Sciences, Sichuan University for cell
imaging measurement.

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