MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATES
This article reports equilibrium and kinetic data for three fluorescein dyes containing the nitro group in the residue of the phthalic acid, 3'-, 4'-, and 5'-nitrofluorescein, in water and 50 mass % aqueous ethanol. These compounds, particularly the recently synthesized 3'-nitrof...
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| author | Kharchenko, Daria Shekhovtsov, Sergiy Cheipesh, Tetiana Mchedlov-Petrossyan, Mykola |
| author_facet | Kharchenko, Daria Shekhovtsov, Sergiy Cheipesh, Tetiana Mchedlov-Petrossyan, Mykola |
| author_institution_txt_mv | [
{
"author": "Daria Kharchenko",
"institution": "PhD student, Department of Physical Chemistry, Chemical Faculty, V.N. KarazinKharkivNational University"
},
{
"author": "Sergiy Shekhovtsov",
"institution": "Senior researcher, Department of Physical Chemistry, Chemical Faculty, V.N. KarazinKharkivNational University"
},
{
"author": "Tetiana Cheipesh",
"institution": "Associate Professor, Department of Physical Chemistry, Chemical Faculty, V.N. KarazinKharkivNational University"
},
{
"author": "Mykola Mchedlov-Petrossyan",
"institution": "V. N. Karazin Kharkiv National University, Svobody sq., 4, Kharkiv, 61022, Ukraine"
}
] |
| author_sort | Kharchenko, Daria |
| baseUrl_str | https://ucj.org.ua/index.php/journal/oai |
| collection | OJS |
| datestamp_date | 2026-07-22T08:23:55Z |
| description | This article reports equilibrium and kinetic data for three fluorescein dyes containing the nitro group in the residue of the phthalic acid, 3'-, 4'-, and 5'-nitrofluorescein, in water and 50 mass % aqueous ethanol. These compounds, particularly the recently synthesized 3'-nitrofluorescein, were less considered polyprotic acids than many other fluorescein dyes. The dyes behave as diprotic acids, which can also be protonated: H3R+⇌ H2R ⇌ HR–⇌ R2–. The dissociation constants in water and 50 mass % aqueous ethanol were obtained in buffer solutions of diluted HCl using the spectrophotometric method accompanied by a potentiometric determination of pH at an ionic strength of 0.05 M and 25.0 ± 1 °C. The dye concentrations in the working solutions were, as a rule, within the range of (5–10)×10–6 M. The thermodynamic values of the dissociation constants were calculated using the Debye–Hückel equation, the second approach, for ionic activity coefficients. The absorption spectra in the visible region of the individual molecular and ionic forms were determined for the above-mentioned compounds and the ester of 3'-nitrofluorescein. For the last, the dissociation constants were determined in aqueous ethanol. Whereas in water the fraction of the zwitterionic tautomer of nitrofluoresceins increases as compared with the parent compound, in aqueous ethanol, the colorless lactone predominates. The microscopic dissociation constants were determined and their values are in line with the influence of the electrophilic nitro group. The alkaline hydrolysis of the nitrofluorescein diacetates was investigated in the two aforementioned solvents. The obtained results allowed us to single out the rate constants of the two-step kinetics of the hydrolysis of the diacetyl derivatives of nitrofluoresceins. |
| doi_str_mv | 10.33609/2708-129X.90.9.2024.3-18 |
| first_indexed | 2025-09-24T17:43:58Z |
| format | Article |
| fulltext |
3
UDK 544.363 + 544.4 + 547.815.1 doi: 10.33609/2708-129X.90.9.2024.3-18
MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE
EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATES
D. V. Kharchenko, S. V. Shekhovtsov, T. A. Cheipesh, N. O. Mchedlov-Petrossyan*
V. N. Karazin Kharkiv National University,
4 Svoboda sq., 61022 Kharkiv, Ukraine
*e-mail: mchedlov@karazin.ua
This article reports equilibrium and kinetic data for three fluorescein dyes containing the nitro
group in the residue of the phthalic acid, 3'-, 4'-, and 5'-nitrofluorescein, in water and 50 mass %
aqueous ethanol. These compounds, particularly the recently synthesized 3'-nitrofluorescein, were
less considered polyprotic acids than many other fluorescein dyes. The dyes behave as diprotic acids,
which can also be protonated: H3R
+ ⇌ H2R ⇌ HR– ⇌ R2–. The dissociation constants in water and
50 mass % aqueous ethanol were obtained in buffer solutions of diluted HCl using the spectropho-
tometric method accompanied by a potentiometric determination of pH at an ionic strength of 0.05
M and 25.0 ± 1 °C. The dye concentrations in the working solutions were, as a rule, within the range
of (5–10)×10–6 M. The thermodynamic values of the dissociation constants were calculated using the
Debye–Hückel equation, the second approach, for ionic activity coefficients. The absorption spectra
in the visible region of the individual molecular and ionic forms were determined for the above-men-
tioned compounds and the ester of 3'-nitrofluorescein. For the last, the dissociation constants were
determined in aqueous ethanol. Whereas in water the fraction of the zwitterionic tautomer of nitro
fluoresceins increases as compared with the parent compound, in aqueous ethanol, the colorless lac-
tone predominates. The microscopic dissociation constants were determined and their values are in
line with the influence of the electrophilic nitro group. The alkaline hydrolysis of the nitrofluorescein
diacetates was investigated in the two aforementioned solvents. The obtained results allowed us to
single out the rate constants of the two-step kinetics of the hydrolysis of the diacetyl derivatives of
nitrofluoresceins.
Keywords: Nitrofluorescein; acid-base dissociation; water-ethanol binary solvent; absorption
spectra; dissociation constants; tautomerism; diacetylfluorescein; two-step alkaline hydrolysis; rate
constants.
INTRODUCTION. This paper continues
the research of alkaline hydrolysis of diacetyl-
fluorescein [1]. Here, we present the study of
diacetates of three nitrofluoresceins with the
NO2 group in the phthalic acid residue in wa-
ter and 50 mass % aqueous ethanol. To better
understand the kinetic data, we examined the
acid-base and tautomeric equilibria of these
nitrofluoresceins and an ester as model com-
pound in the same solvent.
4 ISSN 2708-129X. Укр. хім. журн., 2024
MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATESPHISICAL CHEMISTRY
Scheme 1. The molecular structures of nitrofluoresceins, their diacetates, and esters.
The dissociation constants of 3'-, 4'-, and
5'-nitrofluorescein were determined via the
spectrophotometric method. The hydrolysis of
fluorescein diacetates and similar esters has
been numerously studied in the literature [2–
4]. Usually, a rate constant of the total hydro
lysis was determined without dividing it into
two separate constants of stepwise hydroly-
sis. We have developed an approach based
on the knowledge of the spectrum of the sin-
gle-charged fluorescein anion, which allowed
calculating the first and second rate constant
[1, 5]. Now, we decided to further this proce-
dure by involving nitrofluoresceins. To the best
of our knowledge, the acid-base and tautomer-
ic equilibria of these dyes in aqueous media
have not been studied so far. Therefore, a study
of their protolytic equilibrium in itself is essen-
tial for understanding the behavior of fluores-
cein dyes in solutions.
The dyes behave as diprotic acids, which
can also be protonated, Equations (1–3).
H3R
+ ⇌ H2R + H+, Ka0 (1)
H2R ⇌ HR– + H+, Ka1 (2)
HR– ⇌ R2– + H+, Ka2 (3)
EXPERIMENT AND DISCUSSION OF
THE RESULTS. The synthesis of the dyes and
their diacetates was described in our previous
publications [6, 7]. Hydrochloric, acetic, phos-
phoric, diethylbarbituric acids and analyti-
cal grade borax were used to create different
pH values. The standard aqueous solution of
NaOH was prepared from a saturated solution
using CO2-free water and kept protected from
the atmosphere. Besides 96% aqueous ethanol,
acetonitrile (Sigma-Aldrich, for HPLC, gradi-
ent grade, ⩾ 99.9%) was used to prepare the
stock solutions of the dyes. The equilibrium
and kinetic experiments were run on a Hi-
tachi U-2000 spectrophotometer at 25.0±1 °C.
Because of the limited solubility in water, the
stock solutions of the dyes were prepared in ace-
tonitrile, adding 0.013 M diazabicyclo[5.4.0]
undec-7-ene, DBU. The dye concentrations in
the working solutions were within the range
of (5–10)×10–6 M unless otherwise specified;
hereafter, 1 M = 1 mole dm–3. The pH values
were determined using a glass electrode in a
cell with a liquid junction. The glass electrode
was calibrated with standard buffer solutions
(pH 1.68, 4.01, 6.86, and 9.18 at 25 °C). When
working in 50 mass % ethanol, the instrumen-
tal pH values, pHinstr, were corrected according
to the literature data: pH = pHinstr – 0.20 [8].
The limiting alkaline forms of the dyes were
obtained in diluted NaOH and, in some cas-
es, with DBU (Merck, for synthesis). For each
system, the number of solutions with differ-
ent pH was 25 to 32. The CLINP chemometric
program and wavelengths from 430 to 510 nm
5https://ucj.org.ua
D. V. Kharchenko, S. V. Shekhovtsov, T. A. Cheipesh, N. O. Mchedlov-Petrossyan UCJ № 9 / Vol. 90
with a step of 1 nm were used for calculations
[7, 9]. The ionic strength I = 0.05 M was main-
tained by sodium chloride of analytical grade.
In some acidic solutions, e.g., with pH in water
below 1.3, the ionic strength in HCl solutions
was higher. The hydrolysis kinetics of fluores-
cein dyes was studied as described previous-
ly [1]. The visible spectra were recorded from
400 to 600 nm during either 29 or 40 s for fas
ter and slower reactions, respectively. The time
of adding the buffer was considered as the mo-
ment of beginning the reaction.
The visible absorption spectra at different
pH in water and the dependences of absor
bance on pH are exemplified in Figures 1
and 2, respectively.
Fig. 1 – The absorption spectra of 3'-nit
rofluorescein in aqueous solutions, 25 °C, at
different pH values; The dye concentration
1.08×10–5 M; I = 0.05 M except forsolutions
with pH < 1.3. The pH values are given in the
activity scale down to pH 1.3; the concentra-
tion scale (–logarithm of HCl concentration)
was used below this limit.
Fig. 2 – The dependence of absorbance
of 3'-nitrofluorescein on pH at wavelengths
448 nm (1), 470 nm (2), and 501 nm (3);
25 °C, I = 0.05 M, except the solutions with
pH below 1.3.
6 ISSN 2708-129X. Укр. хім. журн., 2024
MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATESPHISICAL CHEMISTRY
The results are presented in Table 1. The
thermodynamic values of the dissociation con-
stants were calculated using the Debye – Hück-
el equation, the second approach, for ionic ac-
tivity coefficients. The values of activity coef-
ficients in water are 0.822 and 0.457 for single
and double-charged ions, respectively. The ab-
sorption spectra of the individual equilibrium
forms of the dyes are presented in Figures 3–5.
The spectra of the H3R
+ and R2– forms were
measured directly in 1.0 M HCl and 0.042 M
NaOH solutions, respectively, while those of
H2R and HR– were singled out from the expe
rimental data jointly with calculating the dis-
sociation constants.
Table 1.
The parameters of ionic equilibria of nitrofluoresceins in water, 25 °C, I = 0.05 Ma,b
Parameter c 3'-Nitrofluorescein d 4'-Nitrofluorescein 5'-Nitrofluorescein
pKa0 1.06±0.01 (0.98) 1.86±0.01 (1.78) 1.87±0.02 (1.79)
pKa1 2.82±0.01 (2.90) 3.66±0.01 (3.74) 3.68±0.02 (3.76)
pKa2 6.28±0.01 (6.54) 6.36±0.01 (6.62) 6.54± 0.01 (6.80)
λmax / ε (H3R
+)×10–3 449 nm /55.72 443 nm /59.73 444 nm /60.29
λmax /ε (H2R)×10–3 446 nm /43.89 439 nm /23.86 441 nm /26.56
λmax /ε (HR–)×10–3 459 nm /31.44;
485 nm /29.96
457 nm /34.48
477 nm /33.44
458 nm /35.21
481 nm /33.84
λmax /ε (R2–)×10–3 501 nm /93.1 497 nm /85.65 499 nm /94.03
α (H2Z
±) ≈ 0.8 0.36 0.43
α (H2Q) >0.01 0.11 0.07
α (H2L) ≈ 0.2 0.53 0.50
pK±,COOH ≈ 1.1 2.22 2.14
pK0,OH —d 2.74 2.93
pK1,COOH — d 2.78 2.60
pK1,Z ≈ 2.7 3.30 3.39
Note. a The pKa values in parenthesis are thermodynamic. bThe molar absorptivities, ε, are expressed in M–1cm–1.
cFor fluorescein, the pKa values are 2.22, 4.37, and 6.55; the thermodynamic values are 2.14, 4.45, and 6.80 [10].
dIn this case, the data with pH below 1.3 were not used for calculations.
The spectra of the individual equilibrium
forms of the dyes are presented in Figure 3.
They were singled out from the total body of
spectra by calculating the molar absorptivities
of the forms H2R and HR– together with the
dissociation constants, while those of the ions
H3R
+ and R2– were determined directly at ap-
propriate acidity.
To explain the obtained data, the detailed
scheme of the acid-base and tautomeric equi-
librium should be considered (Scheme 2) [10,
11].
7https://ucj.org.ua
D. V. Kharchenko, S. V. Shekhovtsov, T. A. Cheipesh, N. O. Mchedlov-Petrossyan UCJ № 9 / Vol. 90
Fig. 3. The absorption spectra of equilibrium species of
3’-nitrofluorescein (a), 4’-nitrofluorescein (b), and 5’-nit
rofluorescein (c) in water, 25 °C, I = 0.05 M (NaCl + buffer
mixture), except the spectra of cations. The spectra of the
H3R
+ and R2– forms are measured in limiting acidic and al-
kaline solutions, respectively. The spectra of the H2R and
HR– forms are obtained together with the dissociation con-
stants.
8 ISSN 2708-129X. Укр. хім. журн., 2024
MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATESPHISICAL CHEMISTRY
Scheme 2 – Ionic and molecular structures of fluorescein in solution.
It is well known that the absorption spect
ra of the zwitterionic, H2Z
±, and quinonoidal,
H2Q, tautomers in the visible are similar to
those of the cation, H3R
+ (structure H3Z
+ in
Scheme 1) and anion HR– (structure HQ– in
Scheme 1), respectively [10, 11]. The disso-
ciation of the carboxylic group is known to
lead only to a slight hypsochromic shift of the
band. The lactonic tautomer, H2L, is colorless
due to the sp3-hybridization of the nodal car-
bon atom C9. Using the spectra of the cationic
and anionic forms instead of the correspond-
ing tautomers of the H2R form, respectively,
and taking into account the above-mentioned
slight shift of the absorption maximum resul
ting from the dissociation of the COOH group,
the fractions α of the latter can be estimated
(Table 1). In the case of 3'-nitrofluorescein, the
fraction of the tautomer H2Q is too small to be
determined accurately.
Whereas for the unsubstituted fluorescein,
α (H2Z
±) = 0.22; α (H2Q) = 0.11, and α (H2L)
= 0.67, the stability of the zwitterionic tautom-
er increases in the case of the nitro derivatives.
9https://ucj.org.ua
D. V. Kharchenko, S. V. Shekhovtsov, T. A. Cheipesh, N. O. Mchedlov-Petrossyan UCJ № 9 / Vol. 90
The apparent reason is the increase in the acid-
ity strength of the NO2 group. The 3', 4', and
5' positions correspond to the ortho, meta, and
para positions for the COOH group. The or-
tho effect is so pronounced, that the fractions
of the quinonoid and lactone tautomers of the
3'-nitrofluorescein cannot be estimated accu-
rately enough. For the other two dyes the frac-
tions of the zwitterionic and quinonoidal tau-
tomers can be assessed more reliably (Table 1).
From Scheme 2, Equations (4) and (5) can be
derived.
pKa0 = pK±,COOH + log α(H2Z
±) =
= pK0,OH + log α(H2Q) (4)
pKa1 = pK1,COOH – log α(H2Q) =
= pK1,Z – log α(H2Z
±) (5)
The thermodynamic values of the indi-
ces of the so-called microscopic dissociation
constants for the unsubstituted fluorescein
are as follows: pK±,COOH = 2.80; pK0,OH = 3.10;
pK1,COOH = 3.49; and pK1,Z = 3.79 [10]. The de-
crease in the pK1,COOH values for the 4'-, and
5'-nitrofluoresceins as compared to the parent
dye is 0.71 and 0.89, respectively, while for the
pK±,COOH, the values are 0.58 and 0.66 for the
same dyes. This corresponds to the meta and
para effects of the nitro group in the benzoic
acid and semi-quantitatively agrees with the
standard values σm = 0.71 and σp = 0.78.
The corresponding effects for the pK0,OH
values are 0.36 and 0.17, respectively, and for
pK1,Z, they are 0.49 and 0.40. Thus, despite the
absence of conjugation between the benzoic
and xanthene parts of the fluorescein mole-
cule, the strong electrophilic properties of the
NO2 group, to some extent, affect the acidity
of the OH group. Note that in this case, the in-
fluence of the 4'-substitution displays a more
substantial effect, obviously because it is in
the para position for the nodal carbon atom.
It aligns with the electron transmittancein the
fluorescein molecule, which was earlier stud-
ied in DMSO as a solvent [12].
Interestingly, the decrease in the pK1,Z value
for the 3'-nitrofluorescein, 1.1, is even more
pronounced. Here, the reason may be the steric
factor that changes the angle between the two
parts of the molecule. Note that the decrease
in the pKa2 value, which corresponds to pK2,OH
in Scheme 2, is 0.26; 0.18; and 0 for the 3'-; 4'-;
and 5'-nitrofluorescein, respectively.
The visible absorption spectra at different
pH in 50 mass % aqueous ethanol and the de-
pendences of molar absorptivity on pH are
shown in Figures 4 and 5, respectively.
Fig. 4 – The absorption spectra of 3'-nitroflu
orescein in 50 mass % aqueous ethanol solutions
at different pH, 25°C; The dye concentration
1.31×10–5 M; I = 0.05 M except solutions with pH <
1.3. The pH values are given in the activity scale
down to pH 1.3; the concentration scale (–loga-
rithm of HCl concentration) was used below this
limit.
10 ISSN 2708-129X. Укр. хім. журн., 2024
MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATESPHISICAL CHEMISTRY
Fig. 5 – The dependence of absorbance of
3'-nitrofluorescein on pH in 50 mass % aque-
ous ethanol at wavelengths 454 nm (1), 470 (2),
and 508 nm (3); 25 °C, I =0.05 M, except the
solutions with pH below 1.3.
In Table 2, the results for the equilibria in 50
mass % aqueous ethanol are presented. The re
lative permittivity of this solvent at 25 °C is 49.0
[11]. The thermodynamic values of the disso-
ciation constants were calculated using the De-
bye – Hückel equation, the second approach,
for ionic activity coefficients. The values of ac-
tivity coefficients in water are 0.69 and 0.23 for
single and double-charged ions, respectively.
The spectra of the individual forms of the dyes
are presented in Figure 6.
Table 2.
The parameters of ionic equilibria of nitrofluoresceins in 50 mass % ethanol, I = 0.05 M a,b
Parameter c 3'-Nitrofluorescein 4'-Nitrofluorescein 5'-Nitrofluorescein
pKa0 0.94±0.005 (0.78) 1.12±0.004 (0.96) 0.92±0.008 (0.76)
pKa1 4.33±0.01 (4.49) 5.26±0.01 (5.42) 5.30±0.01 (5.46)
pKa2 6.97±0.01 (7.44) 7.03±0.01 (7.50) 7.12±0.01 (7.59)
λmax /ε (H3R
+)×10–3 454 nm / 49.39 448 nm / 57.474 449 nm / 58.175
λmax /ε (H2R)×10–3 444 nm / 2.631
479 nm / 1.645
459 nm / 1.183;
489 nm / 1.108
455 nm / 1.482
481 nm / 1.123
λ max/ε (HR–)×10–3 448 nm / 31.519
477 nm / 27.880
457 nm / 34.744;
482 nm / 32.195
459 nm / 38.132;
486 nm / 35.741
λmax /ε (R2–)×10–3 508 nm / 101.03 503 nm / 90.206 506 nm / 111.796
α (H2Z
±) 0.018 — —
α (H2Q) 0.058 0.034 0.035
α (H2L) 0.929 0.966 0.965
pK0,OH 2.02 2.43 2.22
pK1,COOH 3.25 3.95 4.00
Note. aThe pKa values in parenthesis are thermodynamic. b The molar absorptivities, ε, are expressed in M–1cm–1.
cFor fluorescein, the thermodynamic pKa values are 0.94, 6.82, and 7.66 [11].
11https://ucj.org.ua
D. V. Kharchenko, S. V. Shekhovtsov, T. A. Cheipesh, N. O. Mchedlov-Petrossyan UCJ № 9 / Vol. 90
Fig. 6. The absorption spectra of equilibrium species of
3’-nitrofluorescein (a), 4’-nitrofluorescein (b), and 5’-nit
rofluorescein (c) in 50 mass % aqueous ethanol; 25 °C, I =
0.05 M (NaCl + buffer mixture), except the spectra of cati-
ons. The spectra of the H3R
+ and R2– forms are measured in
limiting acidic and alkaline solutions, respectively. The spec-
tra of the H2R and HR– forms are obtained together with the
dissociation constants.
12 ISSN 2708-129X. Укр. хім. журн., 2024
MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATESPHISICAL CHEMISTRY
The most striking difference when moving
from water to this binary solvent is the drop
in the molar absorptivity of the neutral form,
H2R. The apparent reason is the substantial
shift of the state of the tautomeric equilibrium
towards the colorless lactone. Following the
above-described approach, the α (H2Q) val-
ue was estimated, while no severe signs of the
zwitterion were observed (Table 2). Only in the
case of 3'-nitrofluorescein, some signs of the
presence of H2Z
± were fixed. On the one hand,
it reflects the instability of the highly polar
tautomer in the organic environment. On the
other hand, it proves the marked increase in
the acidity of the COOH group in the presence
of the NO2 group in the ortho position, which
leads to a small yield of H2Z
± in 50 mass %
aqueous ethanol.
The pK0,OH values for 4'-nitro- and 5'-nitro-
fluorescein decrease by 0.31 and 0.71 units, re-
spectively, when going from water to the aque-
ous ethanol, while the pK1,COOH values increase
by 1.17 and 1.40 units, respectively. Such me-
dium effects are typical for cationic and neut
ral acids, respectively. For fluorescein, these
changes are –0.69 and +1.89, respectively [11].
Further, the difference between pK1,COOH of flu
orescein, and the nitro derivative increases.
For 4'-nitro- and 5'-nitrofluorescein,these val-
ues are 1.43 and 1.38, respectively. Comparing
them with the values for an aqueous solution,
0.71 and 0.89, respectively, one should conclu-
dethat Hammett’s ρ constant increases in non-
aqueous media.
The pK1,COOH values of the nitro derivatives,
3.25, 3.95, and 4.00 (Table 2) are pronouncedly
lower than that of fluorescein (5.38) [11]. This
should be ascribed to the increase in the Ham-
mett’s constant ρ, which is typical for organic
solvents [7].
To better understand the equilibrium in
aqueous ethanol, we determined the dissocia-
tion constants of 3'-nitrofluorescein methyl es-
ter (Scheme 3) synthesized previously [6]. The
solubility of the methyl and ethyl esters in wa-
ter is scarce; therefore the study was performed
in aqueous ethanol.
Scheme 3 – Dissociation of methyl ester of 3'-nitrofluorescein in solution.
The absorption spectra at different pHs are
shown in Figure 7. The equilibrium param-
eters are presented in Table 3. The pKa0value
of this dye corresponds to the pK0,OH of the
parent compound (Table 2); considering the
accumulation of errors in assessing the lat-
ter, the match can be regarded as satisfactory.
This pK0,OH of fluorescein and its ethyl ester in
50 mass % aqueous ethanol are equal to 2.41
and 2.05, respectively [11].
13https://ucj.org.ua
D. V. Kharchenko, S. V. Shekhovtsov, T. A. Cheipesh, N. O. Mchedlov-Petrossyan UCJ № 9 / Vol. 90
Fig. 7. The absorption spectra of 3’-nitrofluorescein methyl ester in 50 mass % ethanol, 25 °C, at diffe
rent pH values; I = 0.05 M except for solutions with pH < 1.3. The pH values are given in the activity scale
down to pH 1.3; the concentration scale (–logarithm of HCl concentration) was used below this limit.
Bold curves represent absorption spectra of equilibrium species.
The difference between the pKa1 = 6.59 of the
methyl ester and pKa2 = 7.44 should be ascribed
to the influence of the negative charge of the
carboxylate group on the dissociation of the OH
group in the resorcinol part, according to the
Bjerrum – Kirkwood – Westheimer equation
[7, 11]. So, the difference between the pKa2of
fluorescein and pKa1 of its ethyl ester is 0.95 [11].
The final part of this communication is
devoted to the kinetics of hydrolysis of the
diacetyl derivatives of the three nitrofluores-
ceins. The two-step hydrolysis (Scheme 4) was
described using the pseudo-first-order rate
constants, k’, at the fixed pH value: k’ = k cOH-.
Table 3
The parameters of equilibria of 3'-nitro
fluorescein methyl ester (Scheme 3) in
50 mass % ethanol a,b
pKa0 1.79±0.01 (1.63)
pKa1 6.43±0.02 (6.59)
λmax / ε (H2R
+)×10–3 456 nm / 63.19
λmax / ε (HR)×10–3 462 nm / 33.34
492 nm / 28.34
λmax / ε (R–)×10–3 514 nm / 107.29
Note. aI = 0.05 M . bThe molar absorptivities, ε, are
expressed in M–1cm–1.
14 ISSN 2708-129X. Укр. хім. журн., 2024
MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATESPHISICAL CHEMISTRY
Scheme 4 – Alkaline hydrolysis of the diacetyl derivative of nitrofluoresceins.
The spectral data are typified in Figure 8.
The normalized spectra demonstrate two types
of colored forms. The main idea is to equating
to the absorption spectrum of the AcR– ion to
that of HR– of the corresponding fluorescein
dye [1, 5]. The spectrum of the final product of
hydrolysis, R2–, was already determined above.
This made it possible to calculate the current
concentrations of the AcR– and R2– forms at
each moment. Then, the current concentra-
tions of the colorless (Ac)2R form can be easily
calculated.
Fig. 8 – Normalized absorption spectra during the hydrolysis of 4'-nitrofluorescein and in water (a)
and in 50 mass % aqueous ethanol (b), 25 °C, I = 0.05 M (NaCl + buffer mixture).
15https://ucj.org.ua
D. V. Kharchenko, S. V. Shekhovtsov, T. A. Cheipesh, N. O. Mchedlov-Petrossyan UCJ № 9 / Vol. 90
Fig. 9 – Dependences of the molar fractions of (Ac)2R (1), AcR– (2), andR2– (3) of 5'-nitrofluorescein in
water (a) and in 50 mass % aqueous ethanol (b), 25 °C, I = 0.05 M (NaCl + borate buffer).
Table 4.
The rate constants of hydrolysis of nitrofluoresceins in solution, 25 °C, I = 0.05 M.
Compound pH k1’, s
–1 k2’, s
–1 k2/k1
Water
Diacetyl fluorescein [1] 9.72 3.5±0.2 4.5±0.3 1.29
Diacetyl 3'-nitrofluorescein 9.67 1.39±0.04 6.72±0.2 4.84
Diacetyl 4'-nitrofluorescein 9.74 4.29±0.13 12.2±0.4 2.85
Diacetyl 5'-nitrofluorescein 9.74 2.44±0.13 9.56±0.5 3.91
50 mass % aqueous ethanol
Diacetyl fluorescein [1] 10.50 13.10±0.4 26.00±0.8 1.98
Diacetyl 3'-nitrofluorescein 10.36 7.16±0.08 27.83±0.3 3.89
Diacetyl 4'-nitrofluorescein 10.38 11.28±0.33 24.69±0.25 2.19
Diacetyl 5'-nitrofluorescein 10.28 8.55±0.19 27.71±0.6 3.24
The dependence of the fractions of the three
forms on time is demonstrated in Figure 9. The
results of the kinetic study are presented in Ta-
ble 4.
A comparison of the rate constants for a
given compound in water and aqueous etha-
nol demonstrates either no significant changes
or deceleration of the hydrolysis in the sec-
16 ISSN 2708-129X. Укр. хім. журн., 2024
MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATESPHISICAL CHEMISTRY
ond solvent. For all the nitro derivatives, the
k2/k1 ratio is higher than that for the parent
dye. However, the reasons are not so clear.
The influence of the nitro group on the k2’
value is similar to that of the Ka2. Although
there is no conjugation between the two parts
of the molecule, the electrophilic properties
of this substituent result in a slight increase in
both Ka2 and k2’. In water, the pKa2s for fluores-
cein, 3'-, 4'-, and 5'-nitrofluorescein are 6.80,
6.54, 6.62, and 6.80, respectively, whereas the
k2’ values after re-calculation to pH 9.74 are
4.93, 7.89, 12.2, and 9.56 s–1, respectively. In
50 mass % ethanol, the correspondent pKa2
values are equal to 7.66, 7.44, 7.50, and 7.59,
while k2’ = 18.8, 27.83, 25.57, and 33.31 s–1 (at
pH 10.36).
The influence of the nitro group on the first
rate constant is less clear, on the one hand, due
to the sp3-hybridization of the nodal carbon
atom C9, and on the other hand, because of
the lack of the equilibrium constant that can
be compared with the k1’ rate constant. In any
case, after reduction to the same pH, the small-
est first rate constant is observed for 3'-nitro
fluorescein, while the largest one – is for the
4'- derivative. Note that 4' is the para position
to the nodal carbon atom.
CONCLUSIONS. The dissociation constants
of 3'-, 4'-, and 5'-nitrofluorescein were deter-
mined in water and 50 mass % aqueous etha
nol. The state of the tautomeric equilibrium of
the molecular forms of the nitrofluoresceins
substantially differs from that of the parent
dye. A feature of the tautomerism of molecular
forms of dyes is a sharp increase in the pro-
portion of zwitterion and destabilization of the
colorless lactone compared to un-substituted
fluorescein. By contrast, the lactone tautomer
predominates in aqueous ethanol. The influ-
ence of the nitro groups on the rate of hydrol-
ysis is relatively small due to the lack of conju-
gation between the two parts of the molecule.
Nevertheless, for all the nitro derivatives, the
k2/k1 ratio is higher than that for the parent dye.
This study was partially supported by
the Ministry of Education and Science
of Ukraine, Ukraine, via grant num
ber 0122U001485 and by the Simons
Foundation, USA, via grant number
1030292.
МОНОНІТРОФЛУОРЕСЦЕЇНИ У ВОДНИХ СЕ-
РЕДОВИЩАХ: КИСЛОТНО-ОСНОВНІ РІВНОВА-
ГИ, ТАУТОМЕРИЗМ ТА ГІДРОЛІЗ ДІАЦЕТАТІВ
Д. В. Харченко, С. В. Шеховцов,
Т. О. Чейпеш, М. О. Мчедлов-Петросян*
Харківський національний університет
імені В. Н. Каразіна,
пл. Свободи, 4, Харків 61022, Україна
*e-mail: mchedlov@karazin.ua
У статті наведено рівноважні та кінетич-
ні дані для трьох флуоресцеїнових барвни-
ків, що містять нітрогрупу в залишку фта-
левої кислоти, 3'-, 4'- і 5'-нітрофлуоресце
їну, у воді та 50 мас.% водному етанолі. Ці
сполуки, зокрема нещодавно синтезований
3'-нітрофлуоресцеїн, вважали менш полі-
протонними кислотами, ніж багато інших
флуоресцеїнових барвників. Барвники по-
водяться як дипротонні кислоти, які також
можуть бути протонованими: H3R
+ ⇌ H2R
⇌ HR– ⇌ R2–. Константи дисоціації у воді
та 50 мас.% водному етанолі отримували в
буферних розчинах розведеної HCl за до-
17https://ucj.org.ua
D. V. Kharchenko, S. V. Shekhovtsov, T. A. Cheipesh, N. O. Mchedlov-Petrossyan UCJ № 9 / Vol. 90
помогою спектрофотометричного методу
з потенціометричним визначенням рН за
іонної сили 0,05 М і 25,0 ±1 °C. Концентрації
барвників у робочих розчинах, як правило,
були в межах (5–10)×10–6 М. Термодинаміч-
ні значення констант дисоціації розрахову-
вали за допомогою рівняння Дебая – Хюк-
келя, другий підхід для коефіцієнтів іонної
активності. Визначено спектри поглинання
у видимій області окремих молекулярних
та іонних форм для вищезазначених спо-
лук та складного ефіру 3'-нітрофлюоресце-
їну. Для останнього також визначали кон-
станти дисоціації у водному етанолі. Якщо
у воді частка цвіттер-іонного таутомерані-
трофлюоресцеїнів збільшується порівняно
з вихідною сполукою, то у водному етанолі
переважає безбарвний лактон. Визначено
мікроскопічні константи дисоціації, зна-
чення яких відповідають впливу електро-
фільної нітрогрупи. Лужний гідроліз діа-
цетатів нітрофлуоресцеїну досліджували у
двох вищезгаданих розчинниках. Отримані
результати дозволили виділити константи
швидкості двостадійної кінетики гідролізу
діацетил-похідних нітрофлуоресцеїнів.
Ключові слова: нітрофлуоресцеїн, кис-
лотно-основна дисоціація, водно-етаноль-
ний бінарний розчинник, спектри погли-
нання, константи дисоціації, таутомерія,
двоступінчатий лужний гідроліз, констан-
ти швидкості.
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Стаття надійшла 25.12.2024.
|
| id | oai:ojs2.1444248.nisspano.web.hosting-test.net:article-687 |
| institution | Ukrainian Chemistry Journal |
| keywords_txt_mv | keywords |
| language | English |
| last_indexed | 2026-07-23T01:12:06Z |
| publishDate | 2024 |
| publisher | V.I.Vernadsky Institute of General and Inorganic Chemistry |
| record_format | ojs |
| resource_txt_mv | ucjorgua/91/66ec40f97b863857aa3e7c6d6de35291.pdf |
| spelling | oai:ojs2.1444248.nisspano.web.hosting-test.net:article-6872026-07-22T08:23:55Z MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATES Kharchenko, Daria Shekhovtsov, Sergiy Cheipesh, Tetiana Mchedlov-Petrossyan, Mykola Nitrofluorescein; acid-base dissociation; water-ethanol binary solvent; absorption spectra; dissociation constants; tautomerism; diacetylfluorescein; two-step alkaline hydrolysis; rate constants. This article reports equilibrium and kinetic data for three fluorescein dyes containing the nitro group in the residue of the phthalic acid, 3'-, 4'-, and 5'-nitrofluorescein, in water and 50 mass % aqueous ethanol. These compounds, particularly the recently synthesized 3'-nitrofluorescein, were less considered polyprotic acids than many other fluorescein dyes. The dyes behave as diprotic acids, which can also be protonated: H3R+⇌ H2R ⇌ HR–⇌ R2–. The dissociation constants in water and 50 mass % aqueous ethanol were obtained in buffer solutions of diluted HCl using the spectrophotometric method accompanied by a potentiometric determination of pH at an ionic strength of 0.05 M and 25.0 ± 1 °C. The dye concentrations in the working solutions were, as a rule, within the range of (5–10)×10–6 M. The thermodynamic values of the dissociation constants were calculated using the Debye–Hückel equation, the second approach, for ionic activity coefficients. The absorption spectra in the visible region of the individual molecular and ionic forms were determined for the above-mentioned compounds and the ester of 3'-nitrofluorescein. For the last, the dissociation constants were determined in aqueous ethanol. Whereas in water the fraction of the zwitterionic tautomer of nitrofluoresceins increases as compared with the parent compound, in aqueous ethanol, the colorless lactone predominates. The microscopic dissociation constants were determined and their values are in line with the influence of the electrophilic nitro group. The alkaline hydrolysis of the nitrofluorescein diacetates was investigated in the two aforementioned solvents. The obtained results allowed us to single out the rate constants of the two-step kinetics of the hydrolysis of the diacetyl derivatives of nitrofluoresceins. V.I.Vernadsky Institute of General and Inorganic Chemistry 2024-10-25 Article Article Physical chemistry Физическая xимия Фізична xімія application/pdf https://ucj.org.ua/index.php/journal/article/view/687 10.33609/2708-129X.90.9.2024.3-18 Ukrainian Chemistry Journal; Vol. 90 No. 9 (2024): Ukrainian Chemistry Journal; 3-18 Украинский химический журнал; ##issue.vol## 90 ##issue.no## 9 (2024): Ukrainian Chemistry Journal; 3-18 Український хімічний журнал; Том 90 № 9 (2024): Ukrainian Chemistry Journal; 3-18 2708-129X 2708-1281 en https://ucj.org.ua/index.php/journal/article/view/687/341 Copyright (c) 2024 Daria Kharchenko, Sergiy Shekhovtsov, Tetiana Cheipesh, Mykola Mchedlov-Petrossyan https://creativecommons.org/licenses/by-nc/4.0 |
| spellingShingle | Kharchenko, Daria Shekhovtsov, Sergiy Cheipesh, Tetiana Mchedlov-Petrossyan, Mykola MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATES |
| title | MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATES |
| title_full | MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATES |
| title_fullStr | MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATES |
| title_full_unstemmed | MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATES |
| title_short | MONONITROFLUORESCEINS IN AQUEOUS MEDIA: ACID-BASE EQUILIBRIA, TAUTOMERISM, AND HYDROLYSIS OF DIACETATES |
| title_sort | mononitrofluoresceins in aqueous media: acid-base equilibria, tautomerism, and hydrolysis of diacetates |
| topic_facet | Nitrofluorescein acid-base dissociation water-ethanol binary solvent absorption spectra dissociation constants tautomerism diacetylfluorescein two-step alkaline hydrolysis rate constants. |
| url | https://ucj.org.ua/index.php/journal/article/view/687 |
| work_keys_str_mv | AT kharchenkodaria mononitrofluoresceinsinaqueousmediaacidbaseequilibriatautomerismandhydrolysisofdiacetates AT shekhovtsovsergiy mononitrofluoresceinsinaqueousmediaacidbaseequilibriatautomerismandhydrolysisofdiacetates AT cheipeshtetiana mononitrofluoresceinsinaqueousmediaacidbaseequilibriatautomerismandhydrolysisofdiacetates AT mchedlovpetrossyanmykola mononitrofluoresceinsinaqueousmediaacidbaseequilibriatautomerismandhydrolysisofdiacetates |