ПРОТОЛІТИЧНА РІВНОВАГА ТЕТРА- І ПЕНТА- НІТРОФЛУОРЕСЦЕЇНУ У БІНАРНОМУ РОЗЧИННИКУ АЦЕТОНІТРИЛ — ДИМЕТИЛСУЛЬФОКСИД (МАСОВЕ СПІВІДНОШЕННЯ 96 : 4)
In this paper, the acid-base and tautomeric equilibria of four nitrofluorescein dyes, 2,4,5,7-tetranitrofluorescein, 2,4,5,7,4’-pentanitrofluorescein, 2,4,5,7,5’-pentanitrofluorescein, and 2,4,5,7-tetranitrofluorescein methyl ester, were studied. As reaction media, a binary solvent acetonitrile – di...
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| Дата: | 2021 |
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| Автори: | , , , |
| Формат: | Стаття |
| Мова: | Англійська |
| Опубліковано: |
V.I.Vernadsky Institute of General and Inorganic Chemistry
2021
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Репозитарії
Ukrainian Chemistry Journal| _version_ | 1871465711089483776 |
|---|---|
| author | Moskaeva, Elena Mosharenkova, Ahina Shekhovtsov, Sergey Mchedlov-Petrossyan, Nikolay |
| author_facet | Moskaeva, Elena Mosharenkova, Ahina Shekhovtsov, Sergey Mchedlov-Petrossyan, Nikolay |
| author_institution_txt_mv | [
{
"author": "Elena Moskaeva",
"institution": "V. N. Karazin Kharkiv National University, Svoboda sq. 4, Kharkiv, 61022, Ukraine"
},
{
"author": "Ahina Mosharenkova",
"institution": "V. N. Karazin Kharkiv National University, Svoboda sq. 4, Kharkiv, 61022, Ukraine"
},
{
"author": "Sergey Shekhovtsov",
"institution": "V. N. Karazin Kharkiv National University, Svoboda sq. 4, Kharkiv, 61022, Ukraine"
},
{
"author": "Nikolay Mchedlov-Petrossyan",
"institution": "V. N. Karazin Kharkiv National University, Svoboda sq. 4, Kharkiv, 61022, Ukraine"
}
] |
| author_sort | Moskaeva, Elena |
| baseUrl_str | https://ucj.org.ua/index.php/journal/oai |
| collection | OJS |
| datestamp_date | 2026-07-22T08:23:46Z |
| description | In this paper, the acid-base and tautomeric equilibria of four nitrofluorescein dyes, 2,4,5,7-tetranitrofluorescein, 2,4,5,7,4’-pentanitrofluorescein, 2,4,5,7,5’-pentanitrofluorescein, and 2,4,5,7-tetranitrofluorescein methyl ester, were studied. As reaction media, a binary solvent acetonitrile – dimethyl sulfoxide (96 : 4 by mass) was used. The acidity scale in this solvent was established previously. The indices of the dissociation constants of the dyes were determined using the spectrophotometric method.
Interpreting the  values requires an understanding of the state of tautomeric equilibria. The behavior of these compounds differs significantly from that of other fluorescein dyes, e.g., halogen derivatives. In the case of the first three compounds, i.e., for dyes with a free carboxylic group, the lactonic structure is predominant not only for the neutral form, but even for the double-charged anion. The single-charged anionic form exists as an equilibrium mixture of a colored (and fluorescent) tautomer and an almost colorless lactone. The fourth compound with esterified carboxylic group exhibits extreme stability in its anionic form. 
Evaluation of the tautomerization constants made it possible to calculate the microscopic equilibrium constants of the stepwise dissociation of dye lactones, k1L and k2L. The consideration of the difference (pk2L – pk1L) allowed estimating the effective relative permittivity of the space between the ionizing groups basing on the Bjerrum – Kirkwood – Westheimer equation. Tautomerism of anions was discussed from the point of view of stabilization of symmetric structures. |
| doi_str_mv | 10.33609/2708-129X.87.05.2021.25-37 |
| first_indexed | 2025-09-24T17:43:39Z |
| format | Article |
| fulltext |
25
УДК 544.3 + 544.7 + 547.633.6 doi: 10.33609/2708-129X.87.05.2021.25-37
PROTOLYTIC EQUILIBRIUM OF TETRA- AND PENTANITROFLUORESCEINS
IN A BINARY SOLVENT ACETONITRILE – DIMETHYL SULFOXIDE
(MASS RATIO 96 : 4)
E.G. Moskaeva, A.V. Mosharenkova, S.V. Shekhovtsov, N.O. Mchedlov-Petrossyan
V. N. Karazin Kharkiv National University, Svoboda sq. 4, Kharkiv, 61022, Ukraine
e-mail mchedlov@karazin.ua
In this paper, the acid-base and tautomeric equilibria of four nitrofluorescein dyes,
2,4,5,7-tetranitrofluorescein, 2,4,5,7,4’-pentanitrofluorescein, 2,4,5,7,5’-pentanitrofluoresce-
in, and 2,4,5,7-tetranitrofluorescein methyl ester, were studied. As reaction media, a binary
solvent acetonitrile – dimethyl sulfoxide (96 : 4 by mass) was used. The acidity scale in this
solvent was established previously. The indices of the dissociation constants of the dyes were
determined using the spectrophotometric method.
Interpreting the apK values requires an understanding of the state of tautomeric equilibria.
The behavior of these compounds differs significantly from that of other fluorescein dyes, e.g.,
halogen derivatives. In the case of the first three compounds, i.e., for dyes with a free carboxyl-
ic group, the lactonic structure is predominant not only for the neutral form, but even for the
double-charged anion. The single-charged anionic form exists as an equilibrium mixture of
a colored (and fluorescent) tautomer and an almost colorless lactone. The fourth compound
with esterified carboxylic group exhibits extreme stability in its anionic form.
Evaluation of the tautomerization constants made it possible to calculate the microscopic
equilibrium constants of the stepwise dissociation of dye lactones, k1L and k2L. The consid-
eration of the difference (pk2L – pk1L) allowed estimating the effective relative permittivity
of the space between the ionizing groups basing on the Bjerrum – Kirkwood – Westheimer
equation. Tautomerism of anions was discussed from the point of view of stabilization of
symmetric structures.
Key words: nitrofluorescein dyes, acetonitrile-DMSO binary solvent, buffer solutions, ab-
sorption spectra, dissociation constants, tautomerism, anionic lactones.
PROTOLYTIC EQUILIBRIUM OF TETRA- AND PENTANITROFLUORESCEINS IN A BINARY SOLVENT
ACETONITRILE – DIMETHYL SULFOXIDE (MASS RATIO 96 : 4)
26 ISSN 2708-129X. Укр. хім. журн., 2021
PHYSICAL CHEMISTRY
INTRODUCTION. Fluorescein and its nu-
merous halogen derivatives are widely used
in chemistry and contiguous fields of science.
Their behavior in solutions was a matter of nu-
merous studies. Nitro derivatives of fluorescein,
however, are much less explored. During past
two decades, some publications devoted to
acid ionization and tautomerism of nitrofluo-
resceins appeared from this laboratory [1–6].
In this paper, we continue our research in this
direction.
The compounds studied are as follows:
2,4,5,7-tetranitrofluorescein, 2,4,5,7,4’-penta
nitrofluorescein, 2,4,5,7,5’-pentanitrofluores-
cein, and 2,4,5,7-tetranitrofluorescein methyl
ester. Except the first one [4, 6], they were syn-
thesized and identified by us; the details will be
published elsewhere.
Scheme 1 – Molecular structure of 2,4,5,7-tetranitrofluorescein and its methyl ester; the pentanitro
fluoresceins bear the NO2 group in 4’ or 5’ positions.
Key words: nitrofluorescein dyes, acetonitrile-DMSO binary solvent, buffer solutions,
absorption spectra, dissociation constants, tautomerism, anionic lactones
INTRODUCTION. Fluorescein and its numerous halogen derivatives are widely used in
chemistry and contiguous fields of science. Their behavior in solutions was a matter of numerous
studies. Nitro derivatives of fluorescein, however, are much less explored. During past two
decades, some publications devoted to acid ionization and tautomerism of nitrofluoresceins
appeared from this laboratory [1–6]. In this paper, we continue our research in this direction.
The compounds studied are as follows: 2,4,5,7-tetranitrofluorescein, 2,4,5,7,4'-
pentanitrofluorescein, 2,4,5,7,5'-pentanitrofluorescein, and 2,4,5,7-tetranitrofluorescein methyl
ester. Except the first one [4, 6], they were synthesized and identified by us; the details will be
published elsewhere.
O
C
O
O
OHHO
NO2NO2
NO2
O2N
O OHO
NO2NO2
NO2
O2N
COOCH3
4/
5/
Scheme 1 – Molecular structure of 2,4,5,7-tetranitrofluorescein and its methyl ester; the
pentanitrofluoresceins bear the NO2 group in 4' or 5' positions.
H2R ⇌ HR− + H+, 𝐾𝐾a1 (1)
HR− ⇌ R2− + H+, 𝐾𝐾a2 (2)
Fluorescein dyes bearing NO2 groups in the xanthenes portion are relatively strong acids.
Therefore, their investigation in terms of apK values should be conducted in a polar weakly
basic solvent, e.g., in acetonitrile. However, even traces of water can distort the results obtained
with this solvent. In order to avoid this interfering factor, a new binary solvent was proposed
recently, namely, acetonitrile with 4 mass % dimethyl sulfoxide [7]. In this solvent, the acidity
– +
2 a1H R HR + H , K
(1)
– 2– +
a 2HR R + H , K (2)
Fluorescein dyes bearing NO2 groups in the
xanthenes portion are relatively strong acids.
Therefore, their investigation in terms of apK
values should be conducted in a polar weak-
ly basic solvent, e.g., in acetonitrile. However,
even traces of water can distort the results ob-
tained with this solvent. In order to avoid this
interfering factor, a new binary solvent was
proposed recently, namely, acetonitrile with
4 mass % dimethyl sulfoxide [7]. In this sol-
vent, the acidity scale ( +
*
H
pa ) was established,
and a set of buffer solutions was proposed. It
was shown that the proton is associated with
the molecules of the protophilic dimethyl sulf
oxide, while other ionic and molecular species
are solvated mainly by acetonitrile. Hence,
this binary solvent can be regarded, as first ap-
proximation, as acetonitrile with lyonium ion
H(DMSO)2
+ [7].
EXPERIMENT AND DISCUSSION OF
THE RESULTS. Acetonitrile was stored several
days over P4O10 and distilled over dehydrated
K2CO3. Water content was 0.015 % as deter-
mined by coulometric Karl Fischer method.
Dimethyl sulfoxide was purified by freezing
and distilled under vacuum; water content
0.023 %. Benzene was purified from water by
P4O10 and distilled; water content ≤ 0.01 %.
Benzoic and salicylic acids were purified by
E.G. Moskaeva, A.V. Mosharenkova, S.V. Shekhovtsov, N.O. Mchedlov-Petrossyan
27https://ucj.org.ua
UCJ № 5 / Vol. 87
sublimation. Potassium benzoate and sodium
hydrosalicylate were purified by re-crystalli-
zation. Picric acid was twice re-crystallized
from ethanol and dried as described previous-
ly [7]. Tetra-n-butylammonium picrate was
prepared by the reaction between picric acid
and tetra-n-butylammonium iodide and puri-
fied by double re-crystallization from metha-
nol. p-Toluenesulfonic acid monohydrate was
re-crystallized from acetonitrile. 98% aqueous
sulfuric acid was of analytical grade. Diaza-
bicyclo[5.4.0]undec-7-ene (DBU) was used
as purchased from Merck. 2,4-dinitrophenol
was re-crystallized from benzene–cyclohexa
ne mixture (1 : 1 by volume). The synthesis
and purification of tetramethylammonium
2,4-dinitrophenolate was described previous-
ly [8]. The purity of 2,6-dinitrophenol, an-
alytical grade, melting point 64 oC. Sodium
2,6-dinitrophenolate was prepared through
the following procedure. 4.00 g of 2,6-dini-
trophenol was solved under heating in 50 %
aqueous ethanol, which contained 2.00 g so-
dium hydroxide. The hot solution was filtrated
through a syringe membrane filter and cooled
to +6 0С. On the next day, the deposit of the
sodium salt of the 2,6-dinitrophenol was fil-
tered, washed with 10 mL of 50 % aqueous eth-
anol and dried. The mass of the dried salt was
4.28 g. After re-crystallization from 50 mL of
50 % aqueous ethanol the mass of the dry salt
was 3.74 g (yield 83.5%).
Absorption spectra of the dyes were run on
a Hitachi U-2000 instrument, against the sol-
vent blanks at 25 oC. The optical path length
was 1.00 cm. Normally, the dye concentrations
were within the range of (0.3 – 4.7)×10–5 M.
Following reagents and buffer systems were
used for acidity variation: picric acid, benzoic
acid – potassium benzoate, salicylic acid – so-
dium salicylate, 2,4-dinitrophenol – sodium
2,4-dinitrophenolate, 2,6-dinitrophenol – so-
dium 2,6-dinitrophenolate, picric acid, tet-
ra-n-butylammonium picrate, p-toluenesul-
fonic acid, sulphuric acid (98 %), and DBU.
For calculations of the +
*
H
pa values, the follow-
ing thermodynamic HApK values of acids were
used as determined previously [7]: 3.30 (pic-
ric), 8.68 (2,4-dinitrophenol), 8.73 (2,6-dini-
trophenol), 9.61 (salicylic), 13.98 (benzoic).
The
2HA
fK − values of homoassociation for the
last two acids are 2.5×102 and 2.2×103 M–1,
respectively, the logarithms of the association
constants of ions, asslogK , for sodium salts are
2.85, 3.46, and 3.46 for 2,4-, 2,6-dinitrophe-
nolate and salicylate, respectively [7]. The rela
tive permittivity of the binary solvent is 36.95
at 25 oC. The ionic activity coefficients of the
buffer components and dyes were calculated
using the Debye–Hückel equation, second ap-
proach; the A and B parameters are 1.58 and
0.48, respectively; the ionic parameter a was
equated to 0.5 nm; Ba = 2.40. Molecular acti
vity coefficients were taken as unity.
Determination of the apK values of the
dyes. The dissociation constants were deter-
mined using the dependence of the absorption
spectra on +
*
H
pa . These data are presented in
Figures 1–5. The +
*
H
pa values were calculated
using the HApK , asslogK , and
2HA
fK − values re-
ported previously [7, 8]. For the picric acid and
2,6-dinitrophenol, the last value is close to zero
owing to the well-known blocking effect, while
for the 2,4-dinitrophenol it is unknown. Three
schemes of +
*
H
pa calculations were used. They
are as follows:
(i) For salicylate, benzoate, 2,6-, and
2,4-dinitrophenolate buffer mixtures, a com-
plete calculation of the equilibrium compo-
sition was made as described earlier [8]. For
PROTOLYTIC EQUILIBRIUM OF TETRA- AND PENTANITROFLUORESCEINS IN A BINARY SOLVENT
ACETONITRILE – DIMETHYL SULFOXIDE (MASS RATIO 96 : 4)
28 ISSN 2708-129X. Укр. хім. журн., 2021
PHYSICAL CHEMISTRY
the 2,6-dinitrophenol, the homoassociation is
not typical. The
2HA
fK − value for 2,4-dinitrophe-
nolate was roughly estimated as 24 M–1, because
in pure acetonitrile it equals 2.5×102 M–1 [9],
whereas in the binary solvent under study the
2HA
fK − values are 1.8 to 8 times lower than in
acetonitrile [8].
(ii) Diluted unbuffered solutions of the pic-
ric acid were used. In this case, the dye concen-
tration was taken into account when calculating
the equilibrium concentrations of all ionic and
molecular species. The ratio of the molecular,
H2R, and ionic, HR–, forms of the given dye was
estimated from the absorption spectra. The sole
exception was the case of the 2,4,5,7-tetrani-
trofluorescein methyl ester, because of the high
concentration of the picric acid.
In the above cases, +
*
H
pa was calculated as
1log[H ] log f+− − .
(iii) The limited acidity and basicity of the
solvent system was created using the H2SO4
and DBU solutions, respectively. In some cas-
es, p-toluenesulfonic acid tetra-n-butylammo-
nium picrate were used.
The apK values of the dyes were re-calculat-
ed to the thermodynamic ones using the acti
vity coefficients f1 and f2 for single and double
charged anions. The value of the ionic strength
was calculated for each working solution.
It did not exceed 0.005 M in buffer solutions
and diluted picric acid. The sole exception was
the 2,4,5,7-tetranitrofluorescein methyl ester
(Figure 1).
In the case of this compound, the dissoci-
ation of the HR form takes place at very low
+
*
H
pa values. The molecular spectrum was avail-
able only in benzene with small H2SO4 addi-
tion. Higher concentrations of the sulfuric acid
and additions of the p-toluenesulfonic acid led
to decolorization, obviously due to hydrolysis
of the ester group and turning to the mother
compound. The appearance of the R– ion in
pure benzene may be caused by small traces of
water (see the Experimental section).
Figure 1 – Absorption spectra of 2,4,5,7-tetrani-
trofluorescein methyl ester (3.1×10–6 M) in the bina-
ry solvent at different picric acid solutions. The spec-
trum of the anionic form R– was obtained in a sali
cylate buffer solution. The spectrum of the entire HR
form was measured in benzene with 0.1 M sulfuric
acid; without such acidifying, some amounts of the
anion R– are present in the solution (dotted curve).
Three wavelengths around the absorption
maximum were used as analytical positions.
Here, the absorption of the molecular form
HR is small. The thermodynamic value a1pK =
1.95±0.19 was obtained from measurements
with 8 different +
*
H
pa values, from 1.45 to
2.35. Here, the utilization of the Debye–Hü
ckel equation is conventional. Note that in this
binary solvent and in entire DMSO, the anion
R– exhibits bright fluorescence.
Much more diluted solutions of picric acid
were used in the case of 2,4,5,7-tetranitrofluo-
rescein (Figure 2a). Here, the neutral form H2R
is almost colorless due to predominance of the
lactonic tautomer, as in other solvents [1,2,4,6].
E.G. Moskaeva, A.V. Mosharenkova, S.V. Shekhovtsov, N.O. Mchedlov-Petrossyan
29https://ucj.org.ua
UCJ № 5 / Vol. 87
Figure 2 – Absorption spectra of 2,4,5,7-tetranitrofluorescein (4.2×10–5 M) in diluted picric acid (a)
and in the salicylate buffer solutions (b). The R2– spectrum was obtained in 0.02 M DBU solution.
The spectrum of the dianion R2– of the dye is
obtained in DBU solution (Figure 2). This ab-
sorption band is shifted to some extent as com-
pared with the spectra in salicylate buffers. This
can be explained by the specific interaction of
this double-charged anion with either DBUH+
cation or salicylic acid molecules. Such interac-
tions may also somewhat contribute to the main
equilibrium. The dependence of absorbance at
522 nm on +
*
H
pa is given in Figure 3.
Figure 3 – Absorption of 2,4,5,7-tetranitroflu-
orescein (4.2×10–5 M) at 522 nm vs. +
*
H
pa . Picric
acid solutions (left-hand side) and salicylate buffers
(right-hand side); 2,6-dinitrophenolate buffer solu-
tions shown with asterisks.
For a fixed wavelength, the following equa-
tion is valid:
+ 2
+ +
* 1 1
a1 1 a1 a2 2HR H R
* 2 * 1 1
a1 1 a1 a2 2H H
( )
A a K f A K K f
A
a a K f K K f
− −
− −
− −
+
=
+ +
. (3)
Here A stands for the absorbance at the
current +
*
H
a value; 2R
A − and
HR
A − are absor
bances under conditions of complete conver-
sion of the dye into the corresponding form.
Here,
2H RA = 0. This equation can be represent-
ed in three forms for calculation of both disso-
ciation constants and the
HR
A − value:
2
+ +
1 1 * 1 1 * 2HR R
a1 1 a2 1 2H H
( ) ( )
A A A A
K f a K f f a
A A
− −− − − − −− −
= +
2
+ +
1 1 * 1 1 * 2HR R
a1 1 a2 1 2H H
( ) ( )
A A A A
K f a K f f a
A A
− −− − − − −− −
= +
(4)
+ +
2 2
1 * 1 * 2HR
a2 2 1 a1 2H H
R R
( )
A A AK f f a K f a
A A A A
−
− −
− −−
= −
− −
+ +
2 2
1 * 1 * 2HR
a2 2 1 a1 2H H
R R
( )
A A AK f f a K f a
A A A A
−
− −
− −−
= −
− −
(5)
+ + 2 +
* 1 1 * 1 1 * 1
a1 1 a2 1 2 a2 1 2HR H H R H
{ 1 ( ) } ( )A A a K f K f f a A K f f a− −
− − − − −= + + −
+ + 2 +
* 1 1 * 1 1 * 1
a1 1 a2 1 2 a2 1 2HR H H R H
{ 1 ( ) } ( )A A a K f K f f a A K f f a− −
− − − − −= + + − (6)
PROTOLYTIC EQUILIBRIUM OF TETRA- AND PENTANITROFLUORESCEINS IN A BINARY SOLVENT
ACETONITRILE – DIMETHYL SULFOXIDE (MASS RATIO 96 : 4)
30 ISSN 2708-129X. Укр. хім. журн., 2021
PHYSICAL CHEMISTRY
If the difference between the dissociation
constants is big enough, the HR
A − value can be
determined directly and only the first items in
the right-hand side of Equations 4 and 5 can be
used for calculation of the constants. But if the
yield of the single-charged anion, HR–, does
not reach 100 % at any +
*
H
pa value, then an
iterative procedure should be used. The meth-
od of calculations was as follows. As first ap-
proximation,
HR
A − was equated to the maximal
absorption in Figure 3. The dissociation con-
stants were calculated under the assumption
that stepwise equilibria are isolated. Then the
HR
A − value was refined using Equation 6, and
the
1
a 2K − and a2K values were successively im-
proved via Equations 4 and 5.
In dilute picric acid solution, the [HR ]−
values were determined from the absorption
spectra. The +
*
H
a values were calculated using
Equation 7, which is derived using the equa-
tions of mass action law for the picric acid,
mass balance, and electroneutrality.
+ +
* 2 * 1
HPic 1 1 HPic HPicH H
( ) { [HR ] } { [HR ]} 0a a K f f K c− − −+ − − − =
+ +
* 2 * 1
HPic 1 1 HPic HPicH H
( ) { [HR ] } { [HR ]} 0a a K f f K c− − −+ − − − =
(7)
First, the 1f value was equated to unity.
Then, the ionic strength was approximately
equated to +
*
H
a , and after evaluation of the 1f
value the calculations were repeated. Finally,
knowing +
*
H
a , [HR ]− , and 1f , the a1K value
was obtained. After five iterations, the results
became stable. The +
*
H
pa values indicated in
Figures 2a and 3 are obtained as a result of the
first iteration.
The calculations of the +
*
H
pa values and
ionic strengths of the buffer mixtures were
performed by Dr. A. V. Lebed in this Depart-
ment using the previously determined equi-
librium constants and calculation procedure
[7]. In Table 1, the data for the salicylate buff-
er solutions are presented. In Tables 2 and 3,
analogous data for the dinitrophenols are gi
ven. These +
*
H
pa s were used in further deter-
minations of the dissociation constants. They
can also be used for other purposes in this bi-
nary solvent.
Table 1
The +
*
H
pa values and ionic strength of buff-
er solutions in acetonitrile with 4 mass %
DMSO: salicylic acid + 0.00487 M sodium
salicylate, 25 oC
Salicylic acid, M +
*
H
pa I, M
0.1733 6.27 0.0041
0.1722 6.28 0.0041
0.1570 6.35 0.0041
0.1037 6.69 0.0038
0.0948 6.76 0.0037
0.0689 7.01 0.0035
0.0584 7.14 0.0034
0.0555 7.18 0.0033
0.0420 7.40 0.0031
0.0118 8.31 0.0021
0.0050 8.86 0.0017
0.0022 9.32 0.0015
E.G. Moskaeva, A.V. Mosharenkova, S.V. Shekhovtsov, N.O. Mchedlov-Petrossyan
31https://ucj.org.ua
UCJ № 5 / Vol. 87
Table 2
The +
*
H
pa values and ionic strength of
2,6-dinitrophenolate buffer solutions in ace-
tonitrile with 4 mass % DMSO, 25 oC
2,6-Dinitro
phenol
Sodium
2,6-dinitro
phenolate
+
*
H
pa I, M
0.3619 0.0510 6.76 0.0049
0.3547 0.0510 6.77 0.0049
0.3521 0.0510 6.78 0.0049
0.2997 0.0048 6.29 0.0012
0.2954 0.0048 6.23 0.0012
0.2881 0.0048 6.31 0.0012
0.1590 0.0048 6.57 0.0012
0.1362 0.0048 6.64 0.0012
0.1317 0.0048 6.65 0.0012
0.0090 0.0040 7.77 0.0011
0.0045 0.0040 8.07 0.0011
Table 3
The +
*
H
pa values and ionic strength of
2,4-dinitrophenolate buffer solutions in ace-
tonitrile with 4 mass % DMSO, 25 oC
2,4-Dinitro
phenol
Sodium
2,4-dinitro
phenolate
+
*
H
pa I, M
0.1086 0.0040 6.30 0.0018
0.0698 0.0010 6.18 0.0006
0.0551 0.0008 6.25 0.0005
0.0549 0.0008 6.26 0.0005
2,4-Dinitro
phenol
Sodium
2,4-dinitro
phenolate
+
*
H
pa I, M
0.0519 0.0005 6.14 0.0004
0.0455 0.0004 6.14 0.0003
0.0449 0.0004 6.15 0.0003
0.0270 0.0040 7.12 0.0014
0.0090 0.0040 7.68 0.0012
0.0045 0.0040 8.01 0.0012
0.0018 0.0040 8.42 0.0011
However, the data obtained with the two
dinitrophenolate buffer systems were conside
red only as supporting information. The rea-
sons are as follows. For the 2,4-dinitrophenol,
the exact value of the homoassociation constant
is unknown, whereas the concentrations of the
2,6-dinitrophenol, which allow receiving neces-
sary acidities, are too high, and some additional
interactions may take place. Only the results ob-
tained with the salicylate buffer solutions were
used for final calculations. Despite the lower
HApK value of 2,6-dinitrophenol (8.73) as com-
pared with that of salicylic acid (9.61), the latter
is more suitable for creating lesser +
*
H
pa values
due to pronounced homoassociation effect.
As a rule, three wavelength around the
absorption maximum of the HR– anion were
used. For instance, the final
HR
A − value at
522 nm (Figure 3) was 2.455. This value was
used to calculate the molar absorptivity of the
single-charged anion.
Spectrophotometric data for the two penta
nitrofluoresceins (Figures 4 and 5) were pro-
cessed in the same way.
table 3
PROTOLYTIC EQUILIBRIUM OF TETRA- AND PENTANITROFLUORESCEINS IN A BINARY SOLVENT
ACETONITRILE – DIMETHYL SULFOXIDE (MASS RATIO 96 : 4)
32 ISSN 2708-129X. Укр. хім. журн., 2021
PHYSICAL CHEMISTRY
Figure 4 – Absorption spectra of 2,4,5,7,4’-pentanitrofluorescein (4.7×10–5 M) in diluted solutions of the
picric acid (a) and in the salicylate buffer solutions (b). The R2– spectrum was obtained in 0.02 M DBU solution.
Figure 5 – Absorption spectra of 2,4,5,7,5’-pentanitrofluorescein (4.4×10–5 M) in diluted solutions of the
picric acid (a) and in the salicylate buffer solutions (b). The R2– spectrum was obtained in 0.02 M DBU solution.
The results are collected in Tables 4 and 5. Table 4
Molar absorptivities of the dyes in acetonitrile – DMSO binary solvent (96 : 4 by mass).
Dye
( )3 1 1
max max, nm 10 ,M cmE − − −×λ
Neutral Monoanion Dianion
2,4,5,7-Tetranitrofluorescein methyl ester 452 (37.9)a 523 (112.0) —
2,4,5,7-Tetranitrofluorescein —b 522 (58.4)c 422 (20.0)
2,4,5,7,4’-Pentanitrofluorescein —b 526 (49.2)c 425 (16.1)
2,4,5,7,5’-Pentanitrofluorescein —b 525 (64.0)c 421 (16.5)
Note. a In benzene with 0.1 M sulfuric acid. b Almost colorless. c Calculated from the equilibrium data jointly
with the dissociation constants.
E.G. Moskaeva, A.V. Mosharenkova, S.V. Shekhovtsov, N.O. Mchedlov-Petrossyan
33https://ucj.org.ua
UCJ № 5 / Vol. 87
Table 5
Indices of the thermodynamic dissociation constants of the dyes in acetonitrile – DMSO
binary solvent (96 : 4 by mass).
Dye a1pK a2pK
a2pK – a1pK
2,4,5,7-Tetranitrofluorescein methyl ester 1.95± 0.19 — —
2,4,5,7-Tetranitrofluorescein 4.49± 0.08 6.48± 0.17 1.99
2,4,5,7,4’-Pentanitrofluorescein 4.02± 0.03 6.58± 0.10 2.56
2,4,5,7,5’-Pentanitrofluorescein 4.11± 0.08 6.50± 0.16 2.4
Scheme 2 – Dissociation equilibrium of 2,4,5,7-tetranitrofluorescein methyl ester.
Tautomerism of the dye anions. The neutral
HR and anionic R– forms of the methyl ester of
2,4,5,7-tetranitrofluorescein exists as colored
quinonoidal structures HQ and X–, respective-
ly (Scheme 2).
The esterification of the carboxylic group
makes the formation of lactone impossible.
The absorption spectra were presented above
in Figure 1.
In contrast, the lactone tautomer H2L
(Scheme 3) predominates in the case of neutral
species of the other three dyes. The H2R forms
are almost colorless, and the fraction of mole-
cules existing as quinonoidal tautomer,
2H Qα ,
are out of detection limits in spectra. Moreover,
unlike most fluorescein dyes, 2,4,5,7-tetra
nitro derivatives, both with and without a
nitro group in the phthalic acid residue, are
prone to lactonization even in the dianionic
form R2–. The band maxima of the dianions of
the tetra- and pentanitroderivatives are located
close to the UV portion of the spectrum (Fi
gures 2b, 4b, and 5b; Table 4) because of the
“nitrophenolate” absorption of the lactones L2–
(Scheme 3). Long-wavelength absorption, as
for X– anions in Figure 1, for the dianions R2– is
not observed.
Tautomerism of the dye anions. The neutral HR and anionic R– forms of the methyl ester
of 2,4,5,7-tetranitrofluorescein exists as colored quinonoidal structures HQ and X–, respectively
(Scheme 2).
_
O OO
COOCH3
NO2 NO2
NO2O2N
k
_
H+
O OHO
COOCH3
NO2NO2
NO2O2N
1,OH
HQ X–
Scheme 2 – Dissociation equilibrium of 2,4,5,7-tetranitrofluorescein methyl ester
The esterification of the carboxylic group makes the formation of lactone impossible.
The absorption spectra were presented above in Figure 1.
In contrast, the lactone tautomer H2L (Scheme 3) predominates in the case of neutral
species of the other three dyes. The H2R forms are almost colorless, and the fraction of
molecules existing as quinonoidal tautomer,
2H Q , are out of detection limits in spectra.
Moreover, unlike most fluorescein dyes, 2,4,5,7-tetranitro derivatives, both with and without a
nitro group in the phthalic acid residue, are prone to lactonization even in the dianionic form R2–.
The band maxima of the dianions of the tetra- and pentanitroderivatives are located close to the
UV portion of the spectrum (Figures 2b, 4b, and 5b; Table 4) because of the “nitrophenolate”
absorption of the lactones L2– (Scheme 3). Long-wavelength absorption, as for X– anions in
Figure 1, for the dianions R2– is not observed.
PROTOLYTIC EQUILIBRIUM OF TETRA- AND PENTANITROFLUORESCEINS IN A BINARY SOLVENT
ACETONITRILE – DIMETHYL SULFOXIDE (MASS RATIO 96 : 4)
34 ISSN 2708-129X. Укр. хім. журн., 2021
PHYSICAL CHEMISTRY
Scheme 3 – Detailed scheme of protolytic equilibrium of 2,4,5,7-tetranitro dyes with free COOH group.
In contrast, the lactone tautomer H2L (Scheme 3) predominates in the case of neutral
species of the other three dyes. The H2R forms are almost colorless, and the fraction of
molecules existing as quinonoidal tautomer,
2H Q , are out of detection limits in spectra.
Moreover, unlike most fluorescein dyes, 2,4,5,7-tetranitro derivatives, both with and without a
nitro group in the phthalic acid residue, are prone to lactonization even in the dianionic form R2–.
The band maxima of the dianions of the tetra- and pentanitroderivatives are located close to the
UV portion of the spectrum (Figures 2b, 4b, and 5b; Table 4) because of the “nitrophenolate”
absorption of the lactones L2– (Scheme 3). Long-wavelength absorption, as for X– anions in
Figure 1, for the dianions R2– is not observed.
__
O OHHO
C
O
O
NO2NO2
NO2O2N
O OO
C
O
O
NO2 NO2
NO2O2N
_
_
O OO
COOH
NO2 NO2
NO2O2N
k
k 2,L
H+
O OHHO
C
O
O
NO2NO2
NO2O2N
O OO
C
O
O
NO2 NO2
NO2O2N
_
_
O OO
COOH
NO2 NO2
NO2O2N
1,Lk k 2,L
H+
H+
O OHO
COOH
NO2NO2
NO2O2N
1,OH
__
O OHHO
C
O
O
NO2NO2
NO2O2N
O OHO
C
O
O
NO2 NO2
O2N NO2
_
_
O OO
COOH
NO2 NO2
NO2O2N
k
k 2,L
H+
O OHHO
C
O
O
NO2NO2
NO2O2N
_
_
1,Lk k
H+
H+
O OHO
COOH
NO2NO2
NO2O2N
1,OH
H2Q
H2L HL
L2
HX
_
Scheme 3 – Detailed scheme of protolytic equilibrium of 2,4,5,7-tetranitro dyes with free COOH
group
Such deeper colored species appear only for HR– anions of the dyes with four nitro
groups in 2, 4, 5, and 7 positions. The bands resemble that of the monoanion of the ester (Figure
1) but the molar absorptivities are substantially lower, as is seen in Table 4. This allows to
assume that the HR– forms consist of an equilibrium mixture of intensively colored HX–
tautomer and an lactonic anion HL–. Note that the single-charged anions are fluorescent owing to
the presence of the HX– tautomer.
Such deeper colored species appear only for
HR– anions of the dyes with four nitro groups
in 2, 4, 5, and 7 positions. The bands resemble
that of the monoanion of the ester (Figure 1)
but the molar absorptivities are substantially
lower, as is seen in Table 4. This allows to as-
sume that the HR– forms consist of an equilib-
rium mixture of intensively colored HX– tau-
tomer and an lactonic anion HL–. Note that the
single-charged anions are fluorescent owing to
the presence of the HX– tautomer.
If the maxE value of the HX– tautomer of
2,4,5,7-tetranitrofluorescein is equated to that
of the R– (= X–) anion of the methyl ester of
the dye (Table 4), the
HX−α value can be es-
timated as 58.4/112.0 = 0.52. Accordingly,
HL−
α = 1 –
HX−α = 0.48. Analogous evaluations
of the tautomer fractions were made for the
two pentanitrofluoresceins (Table 6).
Knowing these values, the so-called micro-
scopic dissociation constants, k, can be esti-
mated. From the detailed dissociation scheme
(Scheme 3), equations (8)–(10) can be derived:
2,L a2 HL
p p logk K −= + α (8)
21,L a1 H L HL
p p log logk K −= + −α α (9)
21,OH a1 H Q HX
p p log logk K −= + −α α (10)
The 1,Lpk value can be calculated by taking
2H Lα close to unity. The results are collected
in Table 6. The tautomerization constants of
the HX– ⇌ HL– equilibrium are 0.92, 1.27,
and 0.75, respectively. Also, the fractions of
the tautomer H2Q may be estimated. For ex-
ample, if the 1,OHpk value of 2,4,5,7-tetrani-
trofluorescein is equated to that of its methyl
ester, a
2H Qα value of 1.5 310−× follows from
equation (10). Hence, the negligible content
of this tautomer is confirmed in such an in-
direct way.
E.G. Moskaeva, A.V. Mosharenkova, S.V. Shekhovtsov, N.O. Mchedlov-Petrossyan
35https://ucj.org.ua
UCJ № 5 / Vol. 87
Table 6
Fractions of tautomers and the indices of the microscopic dissociation constants.
Dye HX−α
HL−
α 1,Lpk 2,Lpk 2,L 1,Lp pk k− effε
2,4,5,7-Tetranitrofluorescein 0.52 0.48 4.8 6.2 1.4 32
2,4,5,7,4’-Pentanitrofluorescein 0.44 0.56 4.3 6.3 2.0 18
2,4,5,7,5’-Pentanitrofluorescein 0.57 0.43 4.5 6.2 1.7 24
Thus obtained 1,Lpk and 2,Lpk values allow
to evaluate the difference between the stepwise
dissociation of the OH groups of the lactone
H2L, 2,L 1,Lp pk k− (Table 6). The effect can be
explained by the Bjerrum – Kirkwood – Wes-
theimer equation (11), where the term log 4
reflects the statistical factor.
2
A
2,L 1,L
0
24.7p p log 4 0.602
2.303 4 eff eff
e Nk k
RT r r
− = + = +
× πε ε ε
2
A
2,L 1,L
0
24.7p p log 4 0.602
2.303 4 eff eff
e Nk k
RT r r
− = + = +
× πε ε ε
(11)
Here, R, T, and 0ε have their usual mean-
ings, effε is the effective relative permittivity of
the space permeated by the electric field lines,
r is the distance (in nm) between the dissoci-
ating group and the negatively charged sub-
stituent [10]. For more details, see the book by
Vereshchagin [11]. The r = 0.95 nm value for
these dyes was estimated by Dr. V. S. Farafonov
in this Department. Thus estimated effε values
are presented in Table 6. The average value is
25, while the relative permittivity of the sol-
vent equals 36.95. This allows concluding that
the influence of charged group occurs both
through the aromatic molecule and through
the solvent.
Finally, the reasons of the strongly pro-
nounced lactonization of molecules and di-
anions, on the one hand, and commensura-
ble concentrations of HL– and HX– tautomers
should be explained. Electro-deficient nitro
groups reduce the electron density on the no
dal carbon atom thus favoring the intramole
cular acid-base reaction, i.e., lactone forma-
tion. This leads to lactonization even in the
case of dianions. On the other hand, the struc-
ture HL– is an asymmetrical one. In contrast,
the negatively charged chromophore system
of the quinonoidal structure HX– is symmet-
rical, because the image is conventional and
both oxygen atoms are equal [12]. Therefore,
the equipotential charge distribution favors the
stability of the structure. In addition, the pre
sence of DMSO molecules, which are hydrogen
bond acceptors, stabilizes the COOH groups.
CONCLUSIONS. The apK values of 2,4,
5,7-tetranitrofluorescein, 2,4,5,7,4’-, and 2,4,
5,7,5’-pentanitrofluoresceins in acetonitrile
with 4 mass % DMSO are within the range
of 4.0 – 6.6. Whereas the lactone tautomers
predominate in the case of the neutral and
dianionic forms, H2R and R2–, respectively,
the equilibrium concentrations of the colored
quinonoid and almost colorless lactone tauto
mers of the HR- forms of the dyes are commen-
surable; the corresponding tautomerization
constants are close to unity. The microscopic
dissociation constants of lactones and the dif-
ference between their indices, pk2L – pk1L, are
estimated.
PROTOLYTIC EQUILIBRIUM OF TETRA- AND PENTANITROFLUORESCEINS IN A BINARY SOLVENT
ACETONITRILE – DIMETHYL SULFOXIDE (MASS RATIO 96 : 4)
36 ISSN 2708-129X. Укр. хім. журн., 2021
PHYSICAL CHEMISTRY
ACKNOWLEDGEMENTS. The authors
are grateful to Dr. A.V. Lebed and
Dr. V.S. Farafonov for calculations of
some parameters used in this paper, and to
Dr. D.V. Samoylov for the provided sample
of 2,4,5,7-tetranitrofluorescein. This study
was partially supported by the Ministry of
Education and Science of Ukraine via grant
number 0119U002532.
ПРОТОЛІТИЧНА РІВНОВАГА ТЕТРА- І ПЕНТА-
НІТРОФЛУОРЕСЦЕЇНУ У БІНАРНОМУ РОЗ
ЧИННИКУ АЦЕТОНІТРИЛ — ДИМЕТИЛСУЛЬФ
ОКСИД (МАСОВЕ СПІВІДНОШЕННЯ 96 : 4)
О. Г. Москаєва, О. В. Мошаренкова,
С. В. Шеховцов, М. О. Мчедлов-Петросян
Харківський національний університет
імені В. Н. Каразіна, м. Свободи 4, Харків
61022, Україна
e-mail mchedlov@karazin.ua
У цій роботі досліджено кислотно-ос-
новні та тавтомерні рівноваги чотирьох ні-
трофлуоресцеїнових барвників: 2,4,5,7-те-
транітрофлуоресцеїну, 2,4,5,7,4’-пентаніт
рофлуоресцеїну, 2,4,5,7,5’-пентанітрофлуо
ресцеїну та метилового естеру 2,4,5,7-те-
транітрофлуоресцеїну. Як реакційне сере-
довище було обрано бінарний розчинник
асцетонітрил – диметилсульфоксид (96 : 4
за масою). Шкалу кислотності в цьому роз-
чиннику було розроблено раніше. Констан-
ти дисоціації цих барвників було визначено
за допомогою спектрометричного методу.
Інтерпретація значень apK вимагає ро-
зуміння положення тавтомерних рівноваг.
Поведінка цих сполук суттєво відрізня-
ється від такої для інших флуоресцеїнових
барвників, наприклад, галоген-похідних.
У разі перших трьох сполук, тобто для
барвників з вільною карбоксильною гру-
пою, лактонний тавтомер є домінуючим не
тільки для нейтральних форм, але навіть і
для двічі заряджених аніонів. Однозарядні
аніонні форми в розчині існують як рівно-
важна суміш забарвленого (та флуоресцію-
ючого) тавтомера та практично безбарвно-
го лактону. Четверта сполука з естерифі-
кованою карбоксильною групою проявляє
високу стабільність в аніонній формі.
Оцінка констант тавтомерних рівноваг
дозволила розрахувати мікроскопічні кон-
станти дисоціації лактонів барвників, k1L
and k2L. Аналіз різниці (pk2L – pk1L) дозволяє
оцінити ефективну діелектричну проник-
ність простору між групами, що іонізують-
ся, базуючись на рівнянні Б’еррума – Кірк
вуда – Вестхаймера. Тавтомерію аніонів
барвників обговорено з точки зору стабілі-
зації симетричних структур.
Ключові слова: нітрофлуоресцеїнові
барвники, бінарний розчинник ацетоніт-
рил-ДМСО, буферні розчини, спектри по-
глинання, константи дисоціації, тавтоме-
рія, аніонні лактони.
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E.G. Moskaeva, A.V. Mosharenkova, S.V. Shekhovtsov, N.O. Mchedlov-Petrossyan
37https://ucj.org.ua
UCJ № 5 / Vol. 87
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Стаття надійшла 07.06.2021.
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| spelling | oai:ojs2.1444248.nisspano.web.hosting-test.net:article-3112026-07-22T08:23:46Z PROTOLYTIC EQUILIBRIUM OF TETRA- AND PENTANITROFLUORESCEINS IN A BINARY SOLVENT ACETONITRILE – DIMETHYL SULFOXIDE (MASS RATIO 96 : 4) ПРОТОЛІТИЧНА РІВНОВАГА ТЕТРА- І ПЕНТА- НІТРОФЛУОРЕСЦЕЇНУ У БІНАРНОМУ РОЗЧИННИКУ АЦЕТОНІТРИЛ — ДИМЕТИЛСУЛЬФОКСИД (МАСОВЕ СПІВІДНОШЕННЯ 96 : 4) Moskaeva, Elena Mosharenkova, Ahina Shekhovtsov, Sergey Mchedlov-Petrossyan, Nikolay nitrofluorescein dyes, acetonitrile-DMSO binary solvent, buffer solutions, absorption spectra, dissociation constants, tautomerism, anionic lactones. In this paper, the acid-base and tautomeric equilibria of four nitrofluorescein dyes, 2,4,5,7-tetranitrofluorescein, 2,4,5,7,4’-pentanitrofluorescein, 2,4,5,7,5’-pentanitrofluorescein, and 2,4,5,7-tetranitrofluorescein methyl ester, were studied. As reaction media, a binary solvent acetonitrile – dimethyl sulfoxide (96 : 4 by mass) was used. The acidity scale in this solvent was established previously. The indices of the dissociation constants of the dyes were determined using the spectrophotometric method. Interpreting the  values requires an understanding of the state of tautomeric equilibria. The behavior of these compounds differs significantly from that of other fluorescein dyes, e.g., halogen derivatives. In the case of the first three compounds, i.e., for dyes with a free carboxylic group, the lactonic structure is predominant not only for the neutral form, but even for the double-charged anion. The single-charged anionic form exists as an equilibrium mixture of a colored (and fluorescent) tautomer and an almost colorless lactone. The fourth compound with esterified carboxylic group exhibits extreme stability in its anionic form.  Evaluation of the tautomerization constants made it possible to calculate the microscopic equilibrium constants of the stepwise dissociation of dye lactones, k1L and k2L. The consideration of the difference (pk2L – pk1L) allowed estimating the effective relative permittivity of the space between the ionizing groups basing on the Bjerrum – Kirkwood – Westheimer equation. Tautomerism of anions was discussed from the point of view of stabilization of symmetric structures. V.I.Vernadsky Institute of General and Inorganic Chemistry 2021-06-25 Article Article Physical chemistry Физическая xимия Фізична xімія application/pdf https://ucj.org.ua/index.php/journal/article/view/311 10.33609/2708-129X.87.05.2021.25-37 Ukrainian Chemistry Journal; Vol. 87 No. 5 (2021): Ukrainian Chemistry Journal; 25-37 Украинский химический журнал; ##issue.vol## 87 ##issue.no## 5 (2021): Ukrainian Chemistry Journal; 25-37 Український хімічний журнал; Том 87 № 5 (2021): Український хімічний журнал; 25-37 2708-129X 2708-1281 en https://ucj.org.ua/index.php/journal/article/view/311/170 Copyright (c) 2021 Elena Moskaeva, Ahina Mosharenkova, Sergey Shekhovtsov, Nikolay Mchedlov-Petrossyan https://creativecommons.org/licenses/by-nc/4.0 |
| spellingShingle | Moskaeva, Elena Mosharenkova, Ahina Shekhovtsov, Sergey Mchedlov-Petrossyan, Nikolay ПРОТОЛІТИЧНА РІВНОВАГА ТЕТРА- І ПЕНТА- НІТРОФЛУОРЕСЦЕЇНУ У БІНАРНОМУ РОЗЧИННИКУ АЦЕТОНІТРИЛ — ДИМЕТИЛСУЛЬФОКСИД (МАСОВЕ СПІВІДНОШЕННЯ 96 : 4) |
| title | ПРОТОЛІТИЧНА РІВНОВАГА ТЕТРА- І ПЕНТА- НІТРОФЛУОРЕСЦЕЇНУ У БІНАРНОМУ РОЗЧИННИКУ АЦЕТОНІТРИЛ — ДИМЕТИЛСУЛЬФОКСИД (МАСОВЕ СПІВІДНОШЕННЯ 96 : 4) |
| title_alt | PROTOLYTIC EQUILIBRIUM OF TETRA- AND PENTANITROFLUORESCEINS IN A BINARY SOLVENT ACETONITRILE – DIMETHYL SULFOXIDE (MASS RATIO 96 : 4) |
| title_full | ПРОТОЛІТИЧНА РІВНОВАГА ТЕТРА- І ПЕНТА- НІТРОФЛУОРЕСЦЕЇНУ У БІНАРНОМУ РОЗЧИННИКУ АЦЕТОНІТРИЛ — ДИМЕТИЛСУЛЬФОКСИД (МАСОВЕ СПІВІДНОШЕННЯ 96 : 4) |
| title_fullStr | ПРОТОЛІТИЧНА РІВНОВАГА ТЕТРА- І ПЕНТА- НІТРОФЛУОРЕСЦЕЇНУ У БІНАРНОМУ РОЗЧИННИКУ АЦЕТОНІТРИЛ — ДИМЕТИЛСУЛЬФОКСИД (МАСОВЕ СПІВІДНОШЕННЯ 96 : 4) |
| title_full_unstemmed | ПРОТОЛІТИЧНА РІВНОВАГА ТЕТРА- І ПЕНТА- НІТРОФЛУОРЕСЦЕЇНУ У БІНАРНОМУ РОЗЧИННИКУ АЦЕТОНІТРИЛ — ДИМЕТИЛСУЛЬФОКСИД (МАСОВЕ СПІВІДНОШЕННЯ 96 : 4) |
| title_short | ПРОТОЛІТИЧНА РІВНОВАГА ТЕТРА- І ПЕНТА- НІТРОФЛУОРЕСЦЕЇНУ У БІНАРНОМУ РОЗЧИННИКУ АЦЕТОНІТРИЛ — ДИМЕТИЛСУЛЬФОКСИД (МАСОВЕ СПІВІДНОШЕННЯ 96 : 4) |
| title_sort | протолітична рівновага тетра- і пента- нітрофлуоресцеїну у бінарному розчиннику ацетонітрил — диметилсульфоксид (масове співідношення 96 : 4) |
| topic_facet | nitrofluorescein dyes acetonitrile-DMSO binary solvent buffer solutions absorption spectra dissociation constants tautomerism anionic lactones. |
| url | https://ucj.org.ua/index.php/journal/article/view/311 |
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