Journal of Photochemistry & Photobiology, A: Chemistry 413 (2021) 113233

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Journal of Photochemistry & Photobiology, A: Chemistry

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Emission properties of fluorescein in strongly acidic solutions

D. Surzhikova a, M. Gerasimova a, V. Tretyakova a, A. Plotnikov b, E. Slyusareva a, *
a
Siberian Federal University, Svobodny Prospect 79, 660041, Krasnoyarsk, Russia
b
Freiberger Compound Materials GmbH, Am Junger-Löwe-Schacht 5, 09599, Freiberg, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: Experimental determination of the fluorescence quantum yield and spectral profile for a variety of protolytic
Fluorescein forms of fluorescein is challenged by their simultaneous co-existence in aqueous solutions as well as the excited-
Excited-state proton transfer state proton transfer (ESPT). Different methods of fluorescence spectroscopy (both steady-state and time-
Fluorescence
resolved) were applied to investigate aqueous solutions of fluorescein containing sulfuric and hydrochloric
Cation-quinoid equilibrium
Quantum yield
acids (pH < 3.5). The obtained emission spectra were decomposed by Alentsev-Fok method to separate spectral
Excited-state dissociation constant contributions for cationic and neutral quinoid forms. The resolved individual fluorescence spectra were used for
the estimation of the excited-state dissociation constant of the cation-quinoid equilibrium (pka*=-0.5) as well as
fluorescence quantum yield for the quinoid form (0.42). An almost total fluorescence quenching of the cationic
fluorescein was observed in the solutions with high concentration of the hydrochloric acid whereas quantum
yield for this form remains considerable (0.92) in the solutions containing sulfuric acid at similar pH. The ob­
tained results bring new insights to the understanding of the role of the ESPT and counter-ion of the acid in the
relaxation of the excited states of fluorescein in acidic media.

1. Introduction
Fluorescein and its derivatives are widely used as fluorescent probes
[1,2] and acid-base indicators for local pH sensing in biosciences [3–5].
The sensitivity to the environmental acidity is provided by the existence
of a variety of ionic forms of fluorescein including several charged (from
cationic to dianionic) and neutral forms [6,7]. The protolytic state in­
fluences the chromophore, thus the changes in the equilibrium between
the protolytic forms reflect in spectral properties of the dye.
Although most individual protolytic forms in the ground state cannot
be obtained separately in general case the adsorption spectra of fluo­
rescein are considered as well determined [6–9]. The dissociation con­
stants pKa of the cation-neutral, monoanion-neutral and
dianion-monoanion equilibria are 2.25, 4.23, 6.31 respectively [7].
The interpretation of the emission spectra of fluorescein is more
complicated. Only spectra of cation and dianion of fluorescein can be
obtained separately in strongly acidic and alkaline media, respectively,
whereas the determination of individual emission spectra for mono­
anionic and neutral forms is still a challenge due to the a strong spectral
overlap of several forms occuring simultaneously. Moreover, the con­
tributions of protolytic forms to absorption and excitation processes may
differ significantly due to the excited-state proton transfer (ESPT) taking
place between dye and water molecules within the solvation sphere [10,
11]. As the absorption and the following emission relate to different dye
forms, the individual fluorescence quantum yields cannot be determined
correctly using the integral fluorescence intensity and the fraction of
absorbed energy.
Lack of reliable data on individual spectral profiles and the necessity
to employ various approximations for the interpretation of the experi­
mental results lead to a spread of the quantum yield data. Thus, the
values of ~0 [6], 0.39 [12], 0.9-1 [10] were reported previously for the
cationic form whereas the values of ~0 [6,12] and 0.29 [7] for the
neutral. The presence of three tautomers for the neutral form (Fig. 1)
additionally contributes to the interpretation uncertainty. Moreover, the
results obtained for cationic form may strongly depend on the acid used
for the regulation of the acid-base equilibrium in the investigated
system.
A number of attempts was made in order to elucidate electron
properties of the excited states of the protolytic/tautomeric forms not
existing separately. One of them consisted in the simulation the position
of electronic transitions using quantum chemical calculations [13].
Some other approaches were focused on the experimental study of
partial equilibria, existing within a narrow pH range. Thus, the mon­
o-/dianionic equilibrium was investigated thoroughly [14–16]; Orte

* Corresponding author.
E-mail address: ESlyusareva@sfu-kras.ru (E. Slyusareva).

https://doi.org/10.1016/j.jphotochem.2021.113233
Received 30 December 2020; Received in revised form 20 February 2021; Accepted 23 February 2021
Available online 26 February 2021
1010-6030/© 2021 Elsevier B.V. All rights reserved.
D. Surzhikova et al. Journal of Photochemistry & Photobiology, A: Chemistry 413 (2021) 113233

6
et al. [17] studied the cationic/neutral protolytic equilibrium of a the concentration of dye after all dilutions was about 2⋅10− mol⋅l− 1.
fluorescein homologue. Unfortunately, individual vibronic spectra
containing crucial information on the emission efficiency for different
2.2. Measurements
fluorescein forms were beyond the consideration in all the publications
mentioned above.
The pH of solutions in the range of pH > 0 were measured directly by
The present study was confined to the lower pH region (<3.5) where
a SevenCompact pH-meter (Mettler Toledo). The solutions with a higher
three protolytic forms of fluorescein may occur: cationic (C), neutral and
acidity were characterized by the concentration of acid. The equivalent
monoanionic (M). Neutral form is supposed to consist of three tauto­
pH value used for the comparison between experimental and simulated
mers: lactone (L), zwitterion (Z) and quinone (Q) (Fig. 1). Both sulfuric
data in highly acidic solutions was calculated considering two-stage
and hydrochloric acids were used to adjust the pH value of dye solutions
dissociation of sulfuric acid and single-stage dissociation of hydrochlo­
because of their importance in biochemical [18] and industrial [19]
ric acid. Taking into account a very complicated interpretation of this
processes where the application of fluorescein-based pH sensors may be
parameter in the concentrated acidic solutions as well as the roughness
especially useful.
of the employed approach, the role of the pH values is rather illustrative
A combination of fluorescence steady-state and time-resolved spec­
in the particular case. It should be noted that all the quantitative esti­
troscopy was successfully applied to obtain individual emission spectral
mations were made using positive pH values only.
profiles for both cationic and quinoid forms of fluorescein, which still
Absorption spectra were measured using a Lambda 35 spectrometer
could not be determined by direct measurements. Fluorescence quan­
(Perkin Elmer). Fluorescence measurements including both steady-state
tum yields and excited-state dissociation constants were estimated for
and time-resolved were conducted on a Fluorolog 3–22 spectrofluo­
these protolytic forms. The role of ESPT and the acidic counter-ion for
rometer (Horiba Jobin Yvon) equipped with lifetime measurement
fluorescence quenching in the investigated system were discussed
accessory. Spectra were obtained with excitation at the wavelength of
thoroughly. The understanding of the relaxation processes involving
400 nm and subsequently corrected for reabsorption effects and the
excited states of fluorescein makes it possible not only to explain the
sensitivity of the registration system.
spread of values in the published data, but also opens up new possibil­
Time-resolved decays were obtained upon excitation with Delta­
ities for using this dye as a fluorescent probe for the local pH sensing in
Diode laser (spectral maximum at 407 nm, pulse width ~ 60 ps) in the
acidic media.
case of solutions with hydrochloric acid and NanoLED diode (spectral
maximum at 453 nm, pulse width <1.3 ns) for solutions with sulfuric
2. Materials and methods
acid. The fluorescence decays were measured at the wavelength of
490 nm with temporal resolution of 28 ps per channel. The further
2.1. Materials
analysis of the obtained data including the deconvolution procedure was
performed by means of DAS6 software (Horiba Jobin Yvon). The
Fluorescein sodium salt C20H10O5Na2 (Fluka) was used for the
measured decays were approximated by a sum of exponential functions
preparation of stock aqueous solution. Hydrochloric and sulfuric acids
using Levenberg-Marquardt algorithm; the fit quality was estimated
(Sigma-Aldrich) were used for pH regulation. Sodium chloride (Fluka)
using weighted residuals and χ2 value. The fluorescence lifetime was
was used to adjust the concentration of chloride-ion at given pH. Finally,
characterized by an average decay time.

Fig. 1. Protolytic/tautomeric forms of fluorescein at pH < 3.5.

2
D. Surzhikova et al. Journal of Photochemistry & Photobiology, A: Chemistry 413 (2021) 113233

All measurements were carried at room temperature in a L-geometry concentration of the photoexcited fluorescein molecules. Further
cell with the cross-section of 10 × 10 mm. computational results are given in the normalized form.

2.3. Decomposition of the measured spectra 2.5. Determination of the wavenumbers of 0-0 transitions, the radiative
lifetime and the fluorescence quantum yield
The measured composite emission spectrum f1(λ) was assumed as a
superposition of two partially overlapping bands with profiles φ1(λ), Wavenumbers of 0-0 transitions were found by a comparison of the
φ1(λ): vibronic profiles of emission and absorption spectra. The intersection
point of the spectral profiles corresponds to the position of the pure
f1(λ)=φ1(λ)+φ2(λ). (1) electron 0-0 transition [22].
The Alentsev-Fok method [20] was used to determine both profiles, The radiative lifetime τrad was calculated according Strickler-Berg
which are unknown in the common case. The procedure requires an relationship [23]:
additional spectrum with different contributions of components (with [ ∫ ∼ ∼ ∫ ∼ ∼ ]− 1
I(ν) d ν ε(ν)d ν
magnitudes a1 and a2 correspondingly). τrad = 2.88⋅10− 9 n2 ∫ ∼− 3 ∼ ∼ ∼ , (8)
ν I(ν) d ν ν
f2(λ)=a1φ1(λ)+a2φ2(λ). (2)
where I(̃ν) and ε( ν ) denote emission spectra and absorption spectra in
∼
The limitation of the method consists in the necessity of the presence
of two spectral regions (one for each component) where the contribution terms of molar extinction coefficient respectively; n is the refractive
of another component is negligible (overlap-free). The coefficients a1 index.
and a2 can be found as f2 (λ)/f1 (λ) ratios on plateaus within the corre­ Fluorescence quantum yield was calculated as a ratio between the
〈 〉
sponding wavelength ranges out of the overlapping area. The band experimentally determined lifetime of the fluorescent state τf and the
profiles can be obtained consequently: calculated τrad value:
〈 〉
f2 (λ) − a2 f1 (λ) τf
φ1 (λ) = , (3) ϕ= . (9)
a1 − a2 τrad

φ2 (λ) = f1 (λ) − φ1 (λ) (4) 3. Results and discussion

Using the separated normalized profiles φ1(λ), φ1(λ) the values of
magnitudes were found by non-linear approximation for each composite 3.1. Absorption and emission spectra
spectrum even if the overlapping area covers the whole wavelength
range. OriginPro software (OriginLab, Northampton, MA) implementing Both the absorption and emission spectra of fluorescein in aqueous
Levenberg-Marquardt algorithm for non-linear approximation was solution reveal a strongly pronounced dependence on the acidity of the
employed for this purpose. solution medium, which can be characterized by pH (Fig. 2a, b) in
particular. Thus, the absorption spectra demonstrate growth of their
intensity with the increase of the concentration of sulfuric acid in the
2.4. Modeling of pH-dependend equilibrium between protolytic forms
solution whereas their profile and maximum position remain nearly the
same. The evolution of the emission spectra is more sophisticated
Dissociation constants (kA− B ) determines the quantitative pairwise
exhibiting both changes in the shape and a significant (up to 50 nm) blue
relationships between the equilibrium concentrations of protolytic
shift of the spectral maximum. No any remarkable difference in spectra
forms A and B of fluorescein in the ground state [7]. Thus, the concen­
was revealed for fluorescein solutions if hydrochloric acid was used
tration of each form can be found by solving of the following linear
instead of sulfuric acid to establish the similar acidity. However, the
system:
⎧ increase of the concentration of hydrochloric acid in the solution leads
⎪
⎪ kQ− M [Q] − [H+ ]⋅[M] = 0 to a significant decrease of the emission intensity.
⎪
⎪
⎨ kC− Q [C] − [H+ ]⋅[Q] = 0 The spectral changes caused by addition of acids were ascribed to the
kZ− M [Z] − [H+ ]⋅[M] = 0 (5) rearrangement of the spectral contributions of different protolytic/
⎪
⎪
⎪ kC− Z [C] − [H+ ]⋅[Z] = 0
⎪ tautomeric forms of fluorescein [7]. To resolve the contribution of the
⎩
kC− L [C] − [H+ ]⋅[L] = 0 individual forms to the emission spectrum the Alentsev-Fok method was
Under consideration of the requirement on the conservation of the applied [18]. By this approach the initial spectra were decomposed into
total concentration of fluorescein [C0] in acidic media (pH < 3.5): two components with the profiles, which are independent of the choice
of the initial spectra f1 (λ), f2 (λ) within the used range of acid concen­
[M] + [Q] + [Z] + [L] + [C] = [C0 ] (6) trations (Fig. 3). The profile with the maximum at 517 nm was exactly
Dissociation constants for the excited state (k∗A− B )
were determined the same for all the solutions, whereas the profile with the maximum
by Förster cycle method [21], which establishes the relationship be­ near 478 nm revealed a negligible difference depending on the acid used
tween the change in the constant value in comparison with the ground for the tuning of the solution acidity (for details see Fig. S1, Table S1).
The attribution of the resolved spectral profiles to certain protolytic/
state one and the difference between the wavenumbers (ν00 ) for pure
∼

tautomeric forms may be challenging. We suppose that the profile φ1

electron 0-0 transitions in neighboring protonated (A) and deprotonated
with maximum at 478 nm corresponds to the cationic form whereas the
(B) forms:
profile φ2 with the maximum at 517 nm and a pronounced shoulder near
∼B
v00 − v00
∼A 550 nm can be attributed to the neutral quinoid form. This conclusion
pkA∗ -B = pkA-B + hc , (7) agrees with the results of quantum-chemical calculations [13] predict­
2.3kT
ing spectral maximum at 475 nm and near 550 nm for cationic and
where k is the Boltzmann constant, T is the temperature, c is the speed of quinoid forms respectively. Other neutral forms (lactonic and zwitter­
light in vacuum, h is Planck constant. The obtained excited-state con­ ionic) are out of our consideration due to either their non-fluorescent
stants were used for the estimation of the concentration of the excited behavior theoretically predicted by a very low oscillator strengths or a
states of dye molecules similarly to Eqs. (5) and (6). It should be noted, crucial disagreement between the predicted spectral properties with the
that the right part of the Eq. (6) in this case contains the total observed results. Further, the proposed attribution of the resolved

3
D. Surzhikova et al.
Fig. 2. Absorption (a) and emission (b) spectra of fluorescein in solutions with sulfuric acid as pH regulator.
Fig. 3. Spectral profiles of individual protolytic forms of fluorescein decom­
posed from emission spectra of fluorescein in solutions with hydrochloric acid
(solid lines) and sulfuric acid (dashed lines) by Alentsev-Fok method.
spectral profiles to C- and Q-forms of fluorescein will be additionally
verified by a comparison of the simulations with the experimental data.
Taking into account the emission spectral profiles obtained in this
work and the absorption profiles reported previously [7,13] the posi­
tions of 0-0 transitions for protolytic/tautomeric forms of fluorescein
were determined (Table 1, for more details see Fig. S2)
3.2. Fractions of different protolytic forms in ground and excited states
As it follows from Eq. 7 the difference in the 0-0 transition for the
forms distinguishing by their electric charge on one unit reflects in the
different dissociation constant for the ground and excited states.
Depending on the probability ratio for proton transfer and radiative
transition the following situations are possible: (i) preferred emission
from the initial form; (ii) preferred emission from the form appearing
after the proton transfer; (iii) emission from both forms if the proba­
bilities are comparable. According to the previously published results [6,
Table 1
Position of the 0-0 transition and calculated dissociation constants for different protolytic/tautomeric forms of fluorescein upon photoexcitation.
Protolytic/ tautomeric form 0-0 transition, 104 cm− 1
Protolytic equilibrium
C 2.21
Q 2.03
Z 2.21b
b − 0.5
M 2.03
a
Calculated according to (7).
b
The wavenumbers of the 0-0 transitions were obtained supposing similar properties of chromophores for Z- and C-forms as well as Q- and M-forms [7].
4
Journal of Photochemistry & Photobiology, A: Chemistry 413 (2021) 113233
17], the ratio between the probabilities of the proton transfer and
radiative process varies with the pH. Within the investigated pH range
the probability of the photoinduced proton transfer from the cation
amounted ~1011 s− 1 [17], which exceeds typical lifetime for radiative
transitions by 1− 2 orders of magnitude. Thus, we can assume that
protolytic equilibrium in the excited states will be reached within the
lifetime of the latter. In this case, the equilibrium can be described in
terms of the Förster cycle (7).
The calculated dissociation constants of corresponding different in­
dividual protolytic/tautomeric forms of fluorescein in ground (pka) and
excited (pka*) states are given in Table 1.
The excitations changes the ratios between the protolytic/tautomeric
forms in the solution due to the ESPT. In the ground state, the C-form
dominates in the range pH < 2, at pH ~3.5 the fractions of C-, Z-, Q- and
M-forms become comparable (Fig. 4a). Among the discussed neutral
forms, the L-form, despite of its significant contribution, does not absorb
and emit in the visible region [7]. For this reason, the lactone form is not
presented in Fig. 4b. In the excited state, the fraction of Z-form is
negligible in comparison with others. Thus, only three fluorescein forms
(C, Q, M) contributes within the investigated pH range to the emission
which depends both on the fraction of each individual form in the so­
lution at the given pH and the corresponding quantum yield. Only
within a narrow pH range near 1.5 it is possible to observe emission
predominantly from the quinoid form.
The feasibility of the proton transfer can be easily demonstrated by a
comparison of the excitation spectra at pH values below and beyond the
pka for cation-quinoid equilibrium. Thus, the emission spectra demon­
strate contribution of different protolytic forms (Fig. 2b), whereas the
excitation spectra reveal no any significant difference and can be
ascribed to the fluorescein cation entirely (for details see Fig. S3).
The obtained experimental data demonstrates the domination of the
form(s) with the same (or very similar) emission spectra as the quinoid
one within the range 2 < pH < 3.5, whereas the fraction of the cationic
form becomes negligible. At the same time, the simulation results indi­
cate a significant contribution of the excited states of both Q- and M-
forms (Fig. 4b) to the emission spectra. Hence, a very high similarity of
the emission spectral profiles for these forms was assumed that agrees
with the conformity of the chromophore structure [13] for these forms
pka [7] Δpkaa pka*
C-Q 3.20 3.8 − 0.6
Q-M 3.27 0.0 3.3
C-Z 3.01 − 0.1 3.1
Z-M 3.46 3.9
D. Surzhikova et al. Journal of Photochemistry & Photobiology, A: Chemistry 413 (2021) 113233

Fig. 5. Average fluorescence lifetime of fluorescein in solutions with different

concentrations of sulfuric or hydrochloric acids.

acid lifetime remains nearly the same (~3− 3.5 ns) within the whole pH
variation range whereas a significant decrease down to zero values was
observed with the increase of the HCl concentration (for details see
Table S2, Fig. S4).
Within the previously defined pH ranges where the domination of
one single fluorescein form in the emission spectra was observer (pH
~1.5 for quinoid and >6.7 mol⋅l− 1 H2SO4 for cation, Fig. 4b) the esti­
mation of the fluorescence lifetime of individual protolytic forms –
namely, quinoid and cationic – is possible (Table 2). The radiative life­
times and the quantum yield values were calculated as well.
The values of the fluorescence lifetimes determined in the present
work are close to the previously published data for both quinoid and
cation in sulfuric acid solutions (Table 2). It should be noted, that the
type of the acid used for pH regulation may be of crucial importance
influencing the lifetime much stronger than the pH value itself.
The detailed consideration of the determination approach is neces­
Fig. 4. Fractions of fluorescein protolytic/tautomeric forms at different pH in sary for a correct comparison between the values of the quantum yield
ground state (a, simulation) and in excited state (b): the simulated data are obtained in the recent study with the previous results. Thus, the values
shown as solid lines, scatter plot represents the experimental data for sulfuric reported in [6,7,12] were obtained by the use of integrated emission
acid solutions. Numbered points corresponding to strongly acidic solutions are spectra and absorbed energy in comparison with reference (commonly –
shown as empty symbols (1 –6.7 mol⋅l− 1; 2 –3.0 mol⋅l− 1; 3 –1.6 mol⋅l− 1; 4
fluorescein dianion). However, this approach assumes that both ab­
–1 mol⋅l− 1), filled symbols correspond to points within the measurable pH
sorption and emission are attributed to the same protolytic form. The
range. Dashed lines show the approximation of the experimental data (pH > 0)
by a sigmoid function.
impossibility to fulfill this condition if ESPT occurs within the pH range
under consideration leads to a wide spread of the published data.
(Fig. 1).
It should be noted, that the contribution of the quinoid fluorescence Table 2
to the resulting spectra obtained even for the most acidic solutions Emission properties of quinoid and cationic forms of fluorescein.
(3 mol⋅l− 1 HCl and 6.7 mol⋅l− 1 H2SO4) amounts ca. 30 and 13 %
Fluorescence lifetime Quantum yield ϕb
respectively, hence these spectra could not be ascribed to a pure cationic Protolytic/ Radiative 〈 〉
τf , ns
fluorescence. tautomeric lifetime τrad ,
form nsa This Previous This Previous
work studies work studies
3.3. Radiative lifetime, fluorescence lifetime and quantum yield 7.12
2.68 2.97 [7] 0.38 ~0 [6,12]
HCl, pH 1.5
〈 〉 Quinoid (Q) 7.13
The average fluorescence lifetime τf was measured upon excitation
H2SO4, pH 3.00 (pH 1.6) 0.42 0.29 [7]
using two wavelength (407, 453 nm), which belong to the absorption 1.5
bands of C- and Q-forms respectively (Fig. 5). Depending on the pH, the 4.05
variation of the lifetime can be determined by several factors (or their 2 mol⋅l− 1 0.06 3.5− 4.4 [10] <0.01 ~0 [6]
HCl
combinations): (i) the ratio between different fluorescent forms in the Cation (C)
3.85 0.39 [12]
system under consideration; (ii) ESPT; (iii) dynamic and static quench­ 3 mol⋅l− 1 3.55
(5− 10 mol⋅l− 1
0.92 0.9− 1
〈 〉 H2SO4)
ing, etc. The observed dependence of τf on pH represents a compli­ H2SO4 [10]
cated convolution of these factors; the estimation of the contribution of a
calculated according to (8); the refractive index for Q-form was assumed the
each factor is rather a challenge. same as for water, for C-form - as for the corresponding aqueous-acid mixture;
The lifetimes for solutions with different acids (sulfuric or hydro­ the molar extinction coefficients for individual protolytic forms were taken from
chloric) used for pH regulations were similar only for the case of the [7].
maximal pH under consideration (3.5). For solutions containing sulfuric b
calculated according to (9).

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D. Surzhikova et al. Journal of Photochemistry & Photobiology, A: Chemistry 413 (2021) 113233

The results obtained within the scope of the recent study do not
suffer from the methodology drawback described above: they employ
the data on fluorescence lifetimes and spectral profiles for individual
protolytic forms (quinoid and cationic) in different states separately. A
similar approach based on (9) was reported by Martin et al. [10] for the
cationic form only. A high emission efficiency (0.92) with observed for
the cationic form of fluorescein in sulfuric acid solutions, that conforms
to the previous results [10]. The determined value is comparable with
the one for the dianionic form in alkaline solutions. The quantum yield
of the quinoid form was lower, but still high enough amounting ca. 0.4.
Considering the known quantum yields and spectral profiles for C-
and Q-forms (φ1 and φ2 respectively) their contributions to the
measured emission spectrum were calculated for solutions with different
pH (Fig. 4b, scatter plots; for further details on the calculation procedure
see Fig. S5, Table S3). The intersection point of the sigmoid curves
extrapolation for C- and Q-forms at the measured pH data (pH > 0) at ~
Fig. 6. Emission spectra of fluorescein in a 1 mol⋅l− 1 sulfuric acid solution with
-0.5 corresponds to the excited-state dissociation constant of cation- different concentrations of sodium chloride. Insert: Stern-Volmer plot of fluo­
quinoid equilibrium (pka*), which is in a good agreement with the rescence quenching by Cl− .
calculated value of -0.6 (Table 1). The observed deviation from the
calculated value should be considered as satisfactory taking into account 4. Conclusions
uncertainty of the determination of the position of 0-0 transitions which
significantly limits the estimation precision of the Förster cycle. Thus, Although the spectral properties of fluorescein were intensively
the value of − 1.3 for the constant of cation-quinoid equilibrium was investigated since the very beginning of fluorescence spectroscopy, we
reported in an early publication [24], where the positions of 0-0 tran­ tried to bring some new insights concerning the nature of the excitation
sitions were determined very roughly. In the latter publication [13] relaxation in the protolytic/tautomeric forms existing at equilibrium in
based on combined quantum-chemical calculations and experimental strongly acidic solutions (pH < 3.5). The determination of emission
measurements of spectral properties of protolytic/tautomeric fluores­ spectral profiles of individual forms as well as fluorescence quantum
cein forms, the excited-state dissociation constant was estimated as yields was still rather a challenge due to a significant difference between
− 1.6. However, all the estimated values mentioned above are negative the pH values for ground and excited states at cation-quinoid equilib­
and differ from the corresponding value in the ground state at least by rium. The suggested solution of the problem consisted in the decom­
3.5 indicating a high efficiency of ESPT. position of measured spectra into individual components, which allowed
to estimate both the quantum yields and the excited state dissociation
3.4. Effect of chloride-ions on fluorescein emission constant (pka*=− 0.5). The remarkable difference between the acid-base
properties in ground and excited states (Δpka≥3.5) leads to the rear­
The choice of the acid (sulfuric or hydrochloric) used to adjust the pH rangement of the absorption and emission processes between the pro­
of the fluorescein solution has no any remarkable influence on the tolytic forms. Thus, the major contribution to the absorption was
spectral profiles of both quinoid and cation (Fig. 3). The observed minor ascribed to the cationic form whereas the quinoid was found to be
spectral shift (~1− 2 nm) between the cation spectra measured in responsible for the major emission in the solutions at up to pH 3.2. Both
different strong acidic medium at the same pH can be explained by the cationic and quinoid forms demonstrate high quantum yields (0.92 and
difference in dielectric constant of the solutions. The ratio between 0.42 respectively) in the solutions with sulfuric acid. However, hydro­
cation and quinoid forms in the excited state at the given pH remains the chloric acidfig employed to shift the equilibrium in the solutions also
same for both acids. However, the fluorescent lifetime in the solutions acts as an efficient fluorescence quencher (thank to chloride-ions) of the
with a high concentration of hydrochloric acid is significantly shorter cationic form.
than in the solutions of sulfuric acid with the same acidity. Conse­ The obtained results clarifies the mechanisms of the relaxation in
quently, the quantum yield of cation in sulfuric acid solutions is details and may be of importance for the use of fluorescein as probe for
noticeably higher (0.92) than for hydrochloric acid (near zero). various biomedical and industrial applications as well as for environ­
We assumed the presence of the chloride-ion as the major factor mental monitoring.
influencing the fluorescence quenching. The essential role of the
chloride-ion was proved by spectral measurements of the fluorescein
CRediT authorship contribution statement
solution containing 1 mol⋅l− 1 sulfuric acid (pH ~ 0) after addition of
different amounts of NaCl (Fig. 6). In absence of chloride-ions the
D. Surzhikova: Investigation, Formal analysis, Writing - original
fractions of both quinoid and cationic forms are significant (Fig. 4b) and
draft. M. Gerasimova: Methodology, Validation, Visualization, Formal
the average fluorescence lifetime is large enough (~3 ns). A good line­
analysis. V. Tretyakova: Methodology. A. Plotnikov: Software, Visu­
arity of Stern-Volmer plot for fluorescence intensity was observed with
alization, Formal analysis, Writing - review & editing. E. Slyusareva:
the slope of 9.1 mol− 1⋅l (Fig. 6, insert) that gives the quenching constant
Conceptualization, Supervision, Project administration, Writing - review
at 3⋅109 mol− 1⋅l⋅s− 1 taking the fluorescence lifetime (3 ns into consid­
& editing.
eration. The obtained value is close to the upper limit for the constant of
diffusion-controlled quenching at room temperature amounting
1010 mol− 1⋅l s− 1 [25], but still does not exceed it. This indicates the Declaration of Competing Interest
feasibility of the collisional mechanism of fluorescence quenching by
chloride-ions. The observed drastic quenching of fluorescein emission in There are no conflicts to declare.
the solutions with high concentration of hydrochloric acid conforms to
the previous studies, where the fluorescence quenching of organic Acknowledgements
compounds by chlorides was reported [26,27].
This study was supported by the Russian Foundation for Basic
Research, project No. 19-02-00450 and the Ministry of Science and

6
D. Surzhikova et al.
Higher Education of the Russian Federation, project no. FSRZ-2020-
0008.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2021.
113233.
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