Physico-chemical properties of isomeric forms of luminol in aqueous solutions

Article abstract of DOI:10.1016/j.molstruc.2017.10.004

Two luminol isomers, 2,4-diaminoisoindoline-1,3-dione and 8-amino-4-hydroxyphthalazin-1(2H)-one, have been obtained and described, their physical and chemical properties studied. Using UV-spectroscopy and fluorescence data, it has been shown that, in aque

Full text of DOI:10.1016/j.molstruc.2017.10.004

Accepted Manuscript
Physico-chemical properties of isomeric forms of luminol in aqueous solutions
Tatiana A. Skripnikova, Svetlana S. Lysova, Yuriy E. Zevatskii, Leonid V. Myznikov,
Svetlana V. Vorona, Tatiana V. Artamonova
PII:
S0022-2860(17)31335-2
10.1016/j.molstruc.2017.10.004
MOLSTR 24375
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 28 April 2017
Revised Date: 13 September 2017
Accepted Date: 2 October 2017
Please cite this article as: T.A. Skripnikova, S.S. Lysova, Y.E. Zevatskii, L.V. Myznikov, S.V. Vorona,
T.V. Artamonova, Physico-chemical properties of isomeric forms of luminol in aqueous solutions, Journal
of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.10.004.
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ACCEPTED MANUSCRIPT
max=386 nm
max=305 nm
NH+3
NH2
O
NH2
pK2 = 9.85
-H+
O
O
k1
-
NH2
NH
N
N
O
NH2
N
+H+
+H+
O
O
k4
OH-
H+
k3
k2
NH+3
NH2
O
NH2
O
pK2 = 5.05
-H+
pK1 = 2.44
O
-H+
NH
N
NH
NH
+H+
+H+
N
N
O-
OH
OH
max=347 nm; 301 nm
max=350 nm; 294 nm
max=297 nm
k2>k1>>k3>>>k4

Physico-Chemical Properties of Isomeric Forms of Luminol in Aqueous Solutions
ACCEPTED MANUSCRIPT
Tatiana A. Skripnikova*a, Svetlana S. Lysovaa, Yuriy E. Zevatskiia,b,c, Leonid V. Myznikova, Svetlana
V. Voronaa, Tatiana V. Artamonovaa
aSt. Petersburg State University of Industrial Technologies and Design,
191186, St. Petersburg, Bol’shaya Morskaya, 18, Russian Federation
bNOVBYTCHIM Company,
188350, Leningrad Region, Gatchina, Zheleznovodskaya 45, Russian Federation
c Saint Petersburg Electrotechnical University "LETI"
197376, ul. Professora Popova 5, 197376 St. Petersburg, Russian Federation
* Corresponding author. E-mail: t-star07@yandex.ru, Tel: 8-911-231-29-64
Abstract
Two luminol isomers, 2,4-diaminoisoindoline-1,3-dione and 8-amino-4-hydroxyphthalazin-1(2H)-one,
have been obtained and described, their physical and chemical properties studied. Using UV-
spectroscopy and fluorescence data, it has been shown that, in aqueous solutions, 2,4-
diaminoisoindoline-1,3-dione isomerize into 8-amino-4-hydroxyphthalazin-1(2H)-one, with the speed
of transition depending on the pH values. Thermodynamic dissociation constants for the 2,4-
diaminoisoindoline-1,3-dione and 8-amino-4-hydroxyphthalazin-1(2H)-one luminol isomers have
been determined.
Keywords: luminol isomers, UV-vis absorption spectrophotometry, thermodynamic dissociation
constant, fluorescence.
1. Introduction
Over the last 50 years, luminol has become one of the most widely used chemiluminiscent
reagents in biological and analytical chemistry [1]. Luminol is currently used for determining the
heavy metal cations [2], peroxides [3,4] and various organic compounds [5]; it’s also one of the most
important and best-researched chemiluminiscent reagents used in forensic medicine [6,7]. Luminol is
also used in pharmaceutical industry [8].
In the Refs, the structure of luminol is mostly represented as the 5-amino-2,3-dihydro-1,4-
phtalazine-dion (I); it is shown, however, that, in crystalline state, luminol is to be found as the 8-
amino-4-hydroxyphthalazin-1(2H)-one (II) tautomer [9] (Figure 1). However, there seems to be no
data available as to in which form luminol is to be found in the aqueous solution.
H2N
H2N
NH2
O
O
O
NH
N
NH
NH
N
NH2
O
OH
O
III
I
II
Fig. 1. 5-amino-2,3-dihydro-1,4-phtalazine-dion (I), 8-amino-4-hydroxyphthalazin-1(2H)-one (II),
2,4-Diaminoisoindoline-1,3-dione (III).
It’s common knowledge that, depending on the conditions, luminol can be synthesized as one
of two isomers, 8-amino-4-hydroxyphthalazin-1(2H)-one (II) or 2,4-diaminoisoindoline-1,3-dione
(III) (Fig. 1) [10]. Consequently, commercial luminol could be mixture of isomers II and III. So far,
neither the structure nor the physical and chemical properties of the two isomers, nor their equilibria in
aqueous solutions have been properly studied. However, it’s imperative to determine the structure of

these isomers, since III lacks certain important practical properties, such as fluorescence and
ACCEPTED MANUSCRIPT
chemiluminescence [10].
This work presents the research data on the structuring, as well as physic-chemical properties
of the two luminol isomers (II, III), and of their acid-base equilibriums in aqueous solutions.
2. Experimental
2.1 General
1
Commercial luminol was obtained from Vecton. The H and 13C NMR spectral measurements
were performed on a JEOL JNM-ECX 400A (400 and 100 MHz respectively). Leco CHNS-932
analyzer was used for elemental analysis. IR spectra (KBr) were recorded on Shimadzu FTIR-8400S.
Fluorescence spectra were recorded on Panorama 02 Fluorat spectrofluorometer.
The electronic spectra of absorption were recorded on Shimadzu UV-2700 UV-vis
spectrophotometer. Quartz cuvettes (1.001 cm, 5.000 cm) were used during the spectrophotometric
measurements. Temperatures of the investigated solution were monitored by the cuvette holder
Shimadzu TCC-100 Peltier temperature control with the accuracy of ±0.10. All measurements were
carried out at the temperature of 25 °C.
2.2 Obtaining and Identification of the Luminol Isomers II and III
8-amino-4-hydroxyphthalazin-1(2H)-one (II). II was obtained through reprecipitation of
luminol sodium salt, as described in [11]. 1 mol/dm3 NaOH was added to commercial luminol, the
mixture was heated to 80 °C. The resultant solution was neutralized by the solution of 0.5 mol/dm3
HCl to pH≈1. The precipitate was filtered. White crystal powder was obtained, melting point 328-
331˚С. IR (KBr, ν, cm-1): 3457, 3334 (NH2), 3161 (NH), 2917 (OH), 1656 (C=O), 1602 (C=C), 1499,
1492, 1322, 813, 634. 1H NMR (400 MHz, DMSO-d6), δ, ppm: 6.85-6,94 (m, 2H), 7.28 (s, br, 2H),
7.40-7.44 (m, 1H). 13C NMR (100 MHz, DMSO-d6), δ, ppm: 110.12 (C-Ar), 110.96 (C-Ar), 117.06
(C-Ar), 127.13 (C-Ar), 134.51 (C-Ar), 151.16 (C-OH), 152,15 (C-NH2), 161.89 (C=O).
2,4-Diaminoisoindoline-1,3-dione (III). Commercially available luminol was re-crystallized
from the mixture of DMF:isopropanol (1:1). Crystals of bright yellow color were obtained. Melting
point 259-267 °С. IR (KBr, ν, cm-1): 3473, 3330 (NH2), 1752 (C=O), 1706 (C=O), 1637 (C=C), 1482,
1
1417, 1187, 897, 734. H NMR (400 MHz, DMSO-d6), δ, ppm: 4.77 (s, 2H), 6.36 (s, 2H), 6.93-6.88
(m, 2H), 7.34-7.38 (m, 1H). 13C NMR (100 MHz, DMSO-d6), δ, ppm: 107.83 (C-Ar), 111.13 (C-Ar),
121.96 (C-Ar), 131.28 (C-Ar), 135.48 (C-Ar), 146.62 (C-NH2), 167.77 (C=O), 169.12 (C=O).
Anal. Calcd for C8H7N3O2: C, 54.23; H, 3.95; N, 23.72. Found: C, 54.14; H, 4.19; N, 23.85.
2.3 Emission Spectra of the Luminol Isomers II and III
Fluorescence emission spectra were recorded in a quartz cuvette (1.001 cm) in water. Solutions
were excited at suitable wavelength and monitored at 380-570 nm.
2.4 Solubility of the Luminol Isomers II and III
Aqueous solubility of II and III was determined through spectrophotometry. Calibration
curves were used to determine the concentration of luminol isomers in saturated solutions. Solubility
of II in water is 4ꢀ10-4±0.5ꢀ10-4 mol/dm3. Solubility of III in water is 2ꢀ10-4±0.5ꢀ10-4 mol/dm3..
2.5 UV-Vis Spectroscopy of Luminol Isomers II and III in HCl and NaOH Solutions
A 48 hour recording of UV-vis spectra of luminol isomers in 1 M HCl solutions was performed
(Fig. 2), as well as a 48 hour recording in1 M NaOH (Fig. 3); Shimadzu UV-2700 spectrophotometr
was used.

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Fig. 2. UV visible absorption spectra of 2,4-diaminoisoindoline-1,3-dione (III) after the beginning of
the reaction with 1 M HCl solution: (1) – 0.03 hour; (2) 48 hours (a); UV visible absorption spectra of
8-amino-4-hydroxyphthalazin-1(2H)-one (II) in 1 M HCl (b).
Fig. 3. UV visible absorption spectra of 2,4-diaminoisoindoline-1,3-dione (III) after the beginning of
the reaction with 1 M NaOH solution: (1) – 0.3 hour; (2) 48 hours (a); UV visible absorption spectra
of 8-amino-4-hydroxyphthalazin-1(2H)-one (II) in 1 M NaOH (b).
2.6 Concentration Spectrophotometry of the Luminol Isomers II and III
A set of working solutions with varying II and III concentrations was prepared using the
weight dilution method. Sample of the test substance and its solutions were weighted on the analytical
balance MV 210-А with the electronic precision of ±0.1 mg. The density of the aqueous solutions of
the investigated compounds was measured by a DMA-5000M Аnton Paar Density Meter (Austria)
with the electronic precision of ±5ꢀ10-5 g/cm3. Spectra of the luminol forms at various concentrations
were obtained using the Shimadzu UV-2700 spectrometer. The analytical wavelength corresponded to
the largest difference between the optical absorbance of the solutions of the protonated and
unprotonated forms.
A (optical density) – C (concentration of the luminol isomer) dependencies were used as the
primary experimental data for concentration spectrophotometry; they were obtained through recording
spectra of the investigated compound at various concentrations, with the addition of a strong acid
(base). Throughout the experiment, the concentration of the strong electrolyte remained constant and,
as a rule, didn’t exceed 10-3 mol/dm3. In such solutions, with very low ionic strength (I→0), molecular
interactions and associations are negligible, and thus the solutions are close to the ideal; consequently,
the concentration constants are equal to the thermodynamic ones. The obtained spectrophotometric
data from the UV and visible range were processed using Mathcad 14.

Table 1 presents the data for thermodynamic dissociation constants for luminol isomers pKT (II
ACCEPTED MANUSCRIPT
and III), as well as the wavelength λmax, concentrations of the luminol isomer under investigation C,
concentration of strong electrolyte СНХ(YOH), and the interval of variation of the degree of dissociation
α of the investigated compound.
Table 1. Thermodynamic Dissociation Constants of Luminol Isomers II and III
λmax
nm
,
C,
СНХ(YOH)
рКТ,
Luminol
α
mol/dm3
mol/dm3
mol/dm3
1ꢀ10-4 – 3ꢀ10-4
1ꢀ10-4(HCl)
0.01–0.04 2.44±0.15 (1)
0.05–0.15 5.05±0.39 (2)
349.6
II
8ꢀ10-4 – 2ꢀ10-3
(H2O)
385.8 4ꢀ10-4 – 5ꢀ10-4 6.5ꢀ10-4 (NaOH) 0.67–0.88 9.85±0.16 (2)
III
3. Results and discussion
Samples II and III were obtained from commercial luminol through re-crystallization and re-
precipitation, following the procedure detailed in Experimental.
UV spectrum of II in water showed two maximums of optical density, at the wavelengths of
350 nm and 294 nm, spectrum of III in water has optical density maximum at the wavelength of 386
nm (Fig. 4). Comparison of the UV spectra of luminol III and II with the commercial luminol sample
leads to believe that the latter is a mixture of the isomers.
Fig. 4. UV-vis spectra of commercial luminol in aqueous solution (a), II (b) and III (c).
1
13
1
II and III are described using IR and H and C NMR spectroscopy. H NMR III spectrum
has two 4.77, 6.36 ppm signals, corresponding to the NH2 groups signals. 13C NMR III spectrum has
the signals corresponding to the carbonyl carbons with the chemical shifts 167 and 169 ppm. 13C
NMR II spectrum has only one signal corresponding to С=O group 162 ppm, yet a 151 ppm signal
1
appears, that corresponds to Ar-C-OH group. In the H NMR spectrum of compound II there’s only
one signal that corresponds to the amino group, in the 7.28 ppm range.
In the IR spectrum of III, there are two oscillation bands for C=O group in the range of 1752,
1701 cm-1, as well as the intense signals of NH2 group in the range of 3473, 3330 cm-1. In the IR
spectrum of II there’s one oscillation band for C=O group in the range of 1656 cm-1.

The above shows that commercial luminol is a mixture of 2 isomers, 8-amino-4-
ACCEPTED MANUSCRIPT
hydroxyphthalazin-1(2H)-one (II) and 2,4-diaminoisoindoline-1,3-dione (III); the presence of the 5-
amino-2,3-dihydro-1,4-phtalazine-dion (I) tautomer form was not experimentally proved.
Studying the UV-vis spectra of the luminol isomers at different pH values, we have registered
a quick conversion of III into II in the highly acidic environment (pH<1) (k1). In the highly alkaline
environment (pH>10) the conversion of III into II took place as well, but at a slower pace than in the
acid (Fig. 2, 3) (k2).
Our studies of the fluorescent properties of luminol isomers in water have shown that only II is
fluorescent, with the peak value of 426 nm. Fluorescence of III has not been registered. After 168
hours, in aqueous solution of III, a weak fluorescence was registered, with the peak value
corresponding to the fluorescence of II, which testifies to the transition of III into II, the speed of
transition being extremely slow (k3). The constants k1, k2, k3 are the rate constants. The reverse
transition of II into III doesn’t seem to take place.
On the basis of the above, the equilibriums in the aqueous solutions of luminol isomers can be
represented by the following graph (Fig. 5).
λ
max=386 nm
λ
NH2
max=305 nm
NH+3
NH2
O
K
p
2 = 9.85
O
O
-H+
-
NH2
NH
N
N
NH2
N
+H+
+H+
O
O
O
k2
k3
k1
pH>10
pH < 1
NH+3
p
NH2
O
NH2
O
K
2 = 5.05
K
p
1 = 2.44
O
-H+
-H+
NH
N
NH
NH
+H+
+H+
N
N
O-
OH
OH
λ
max=347 nm; 301 nm
λ
max=350 nm; 294 nm
λ
max=297 nm
k1>k2>>k3
Fig. 5. Scheme of the equilibriums in the aqueous solutions of luminol isomers II and III.
There’s an impressive Refs. (ca. 10 papers per year) treating the mechanisms of
chemiluminiscent reactions that involve luminol. However, there’s a much more limited number of
papers treating the acid-base properties of luminol in aqueous solutions [12-15].
Standard methods of obtaining the dissociation constant value can hardly be applied to III,
because of its isomerization into II. That’s why, in order to obtain the pKa value of the luminol
isomers, we used the method where the measurements can be taken promptly, and without the use of
either buffer solutions or highly acid/ highly alkaline environments, the method in question being
concentration spectrophotometry [16, 17]. pKa are shown at Fig. 5 and in Table 1. III typically has
only one dissociation constant, pK2= 9.85, while the value of pK1 cannot be obtained because of the
fast transition of cation III into cation II.
4. Conclusion
Two luminol isomers, 2,4-diaminoisoindoline-1,3-dione (III) and 8-amino-4-
hydroxyphthalazin-1(2H)-one (II), have been obtained from the commercially available luminol, their
1
structuring has been detailed through IR, H and 13C NMR spectroscopy.

Solubility, acid-base properties and fluorescence of luminol isomers in aqueous solutions have
ACCEPTED MANUSCRIPT
been studied.
The data show that only II is fluorescent. We have proved that III passes into II in aqueous
solutions, the speed of transitions depending on the pH values. The reverse transition of II into III has
not been registered.
Thermodynamic dissociation constants for luminol isomers III and II have been obtained.
References
[1] P. Khan, D. Idrees, M. A. Moxley, J. A. Corbett, F. Ahmad, G. von Figura, W. S. Sly, Abdul
Waheed, Md. I. Hassan, Luminol-Based Chemiluminescent Signals: Clinical and Non-clinical
Application and Future Uses, Appl. Biochem. Biotechnol, 173 (2014) 333–355.
DOI: 10.1007/s12010-014-0850-1
[2] Q. Li, J. Wang, Y. He, Selective chemiluminescent sensor for detection of mercury(II) ions using
non-aggregated luminol-capped gold nanoparticles, Sensors and Actuators B, 231 (2016) 64-69.
DOI: 10.1016/j.snb.2016.03.007
[3] Yu. B. Tsaplev, Chemiluminescence determination of hydrogen peroxide, Journal of Analytical
Chemistry, 67 (6) (2012) 506–514.
DOI: 10.1134/S1061934812040028
[4] V. K. Zinchuk, Activity of Aliphatic Diperoxy Acids in the Luminol Chemiluminescence
Reaction, Journal of Analytical Chemistry, 56 (12) (2001) 1105–1108.
DOI: 10.1023/A:1012936900486
[5] C.A. Marquette, L.J. Blum, Applications of the luminol chemiluminescent reaction in analytical
chemistry, Anal. Bioanal. Chem. 385 (3) (2006) 546 – 554.
DOI: 10.1007/s00216-006-0439-9
[6] H. Cui, W. Wang, C.-F. Duan, Y.-P. Dong, J.-Z. Guo, Synthesis, Characterization, and
Electrochemiluminescence of Luminol-Reduced Gold Nanoparticles and Their Application in a
Hydrogen Peroxide Sensor. Chem.-Eur. J. (13) (2007) 6975−6984
DOI: 10.1002/chem.200700011
[7] F.Barni, S.W. Lewis, A. Berti, G.M. Miskelly, G. Lago, Forensic application of the luminol
reaction as a presumptive test for latent blood detection, Talanta 72 (2007) 896–913.
DOI:10.1016/j.talanta.2006.12.045
[8] US 8536171 B2. Method for obtaining 5-amino 2,3-dihydrophthalazine-1,4-dione alkali metal salts
and their use in medicine, Admir Musaevich Abidov, Aleksei Silantievich Ishmuratov, Vitalij Vilkin,
Irina Georgievna Danilova, 17.09.2013
[9] T. Martin, D. Greim, W. Milius, M. Niedemaier, B. Ludescher, J. von Wegerer, W. Brysch, K.
Barwinkel, J. Senker, J. Breu, The Same at a First Glance: The Diffractogram of a New Polymorph of
Anhydrous Sodium Luminolate Almost Perfectly Resembles the Diffraction Trace of an Already
Known Polymorph, journal of Inorganic and General Chemistry, 641 (2) (2015) 332–338.
DOI: 10.1002/zaac.201400604
[10] V.K. Gleu, K, Pfannstiel, Über 3-Aminophthalsäure-hydrazid, Journal fur praktische Chemie N.
B., 146 (1936), 137-150.
DOI: 10.1002/prac.19361460504
[11] T. Martin, J. Fleissner, W. Milius, J. Breu, Behind Crime Scenes: The Crystal Structure of
Commercial Luminol, Cryst. Growth Des. 16 (2016) 3014−3018.
DOI: 10.1021/acs.cgd.6b00425
[12] U. Isacson, J. Kowalewska, G.Weyyermark, Kinetic investigations of reactions between inorganic
chlorine compounds and luminol, J. Inorg. Nucl. Chem. 40 (1978) 1653-1656.
DOI: 10.1016/0022-1902(78)80343-1
[13] L. Erdey, I. Buzas, K. Vigh, Luminol as a fluorescent acid-base indicator, Talanta, 13 (1966) 163
DOI: 10.1016/0039-9140(66)80064-4
[14] G. Merenyi, J. Lind, X. Shen, T.E. Eriksen, Oxidation potential of luminol. Is the autoxidation of
singlet organic molecules an outer-sphere electron transfer? J. Phys. Chem. 94 (1990) 748-752.
[15] K.E. Haapakka, J.J. Kankare, J.A. Linke, Determination of the second acidity constant of
luminol, Anal. Chim. Acta 139 (1982) 379–382

DOI: 10.1016/S0003-2670(01)94020-6
ACCEPTED MANUSCRIPT
[16] S.S. Lysova, T.A. Starikova, Y.E. Zevatskii, Limit of concentration constant of protolytic
equilibrium, Russian journal of general chemistry. 84 (8) (2014) 1634-1635.
DOI: 10.1134/S1070363214080325
[17] T.A. Skripnikova, S.S. Lysova, Y.E. Zevatskii, Determination of the Dissociation Constants of 5-
Substituted 1H-Tetrazoles and Amino Acids by the Concentration Spectrophotometric Method: A
New Approach, J. Chem. Eng. Data 62 (8) (2017) 2400–2405.
DOI: 10.1021/acs.jced.7b00308

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1
1. The structure of two luminol isomers has been detailed through IR, H and 13C NMR
spectroscopy.
2. 2,4-diaminoisoindoline-1,3-dione passes into 8-amino-4-hydroxyphthalazin-1(2H)-one in
aqueous solutions, the speed depending on the pH.
3. Only 8-amino-4-hydroxyphthalazin-1(2H)-one is fluorescent.
4. Thermodynamic dissociation constants for luminol isomers have been obtained.