Synthesis, structure and study of azo-hydrazone tautomeric equilibrium of 1,3-dimethyl-5-(arylazo)-6-amino-uracil derivatives

Article abstract of DOI:10.1016/j.saa.2014.12.027

Azo dyes, 1,3-dimethyl-5-(arylazo)-6-aminouracil (aryl = -C₆H₅ (1), -p-CH₃C₆H₄ (2), -p-ClC₆H₄ (3), -p-NO₂C₆H₄ (4)) were prepared and characterized by UV-vis, FT-IR, ¹H NMR, ¹³C NMR spectroscopic techniques and single crystal X-ray crystallographic analysis. In the light of spectroscopic analysis it evidences that of the tautomeric forms, the azo-enamine-keto (A) form is the predominant form in the solid state whereas in different solvents it is the hydrazone-imine-keto (B) form. The study also reveals that the hydrazone-imine-keto (B) form exists in an equilibrium mixture with its anionic form in various organic solvents. The solvatochromic and photophysical properties of the dyes in various solvents with different hydrogen bonding parameter were investigated. The dyes exhibit positive solvatochromic property on moving from polar protic to polar aprotic solvents. They are fluorescent active molecules and exhibit high intense fluorescent peak in some solvents like DMSO and DMF. It has been demonstrated that the anionic form of the hydrazone-imine form is responsible for the high intense fluorescent peak. In addition, the acid-base equilibrium in between neutral and anionic form of hydrazone-imine form in buffer solution of varying pH was investigated and evaluated the pK_a values of the dyes by making the use of UV-vis spectroscopic methods. The determined acid dissociation constant (pK_a) values increase according to the sequence of 2 > 1 > 3 > 4.

Full text of DOI:10.1016/j.saa.2014.12.027

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, structure and study of azo-hydrazone tautomeric equilibrium
of 1,3-dimethyl-5-(arylazo)-6-amino-uracil derivatives
Diptanu Debnath a, Subhadip Roy a, Bing-Han Li b, Chia-Her Lin b, Tarun Kumar Misra a,
⇑
a Department of Chemistry, National Institute of Technology, Agartala 799046, India
b Department of Chemistry, Chung Yuan Christian University, Chung-Li 320, Taiwan
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Crystal structure of 1,3-dimethyl-5-
phenylazo-6-aminouracil.
Evidenced azo- and hydrazone forms
of azo-derivatives of 6-aminouracil.
Studied solvent effects on the
absorption maxima of the azo-dyes.
Studied photo-physical property of
the azo-dyes.
Evaluated acid-dissociation constant
(pKa) values of the dyes.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 March 2014
Received in revised form 5 December 2014
Accepted 9 December 2014
Available online 29 December 2014
Azo dyes, 1,3-dimethyl-5-(arylazo)-6-aminouracil (aryl = -C6H5 (1), -p-CH3C6H4 (2), -p-ClC6H4 (3), -p-
NO2C6H4 (4)) were prepared and characterized by UV–vis, FT-IR, 1H NMR, 13C NMR spectroscopic tech-
niques and single crystal X-ray crystallographic analysis. In the light of spectroscopic analysis it evi-
dences that of the tautomeric forms, the azo-enamine-keto (A) form is the predominant form in the
solid state whereas in different solvents it is the hydrazone-imine-keto (B) form. The study also reveals
that the hydrazone-imine-keto (B) form exists in an equilibrium mixture with its anionic form in various
organic solvents. The solvatochromic and photophysical properties of the dyes in various solvents with
different hydrogen bonding parameter were investigated. The dyes exhibit positive solvatochromic prop-
erty on moving from polar protic to polar aprotic solvents. They are fluorescent active molecules and
exhibit high intense fluorescent peak in some solvents like DMSO and DMF. It has been demonstrated
that the anionic form of the hydrazone-imine form is responsible for the high intense fluorescent peak.
In addition, the acid-base equilibrium in between neutral and anionic form of hydrazone-imine form
in buffer solution of varying pH was investigated and evaluated the pKa values of the dyes by making
the use of UV–vis spectroscopic methods. The determined acid dissociation constant (pKa) values increase
according to the sequence of 2 > 1 > 3 > 4.
Keywords:
Azo-uracil
Crystal structure
Solvatochromism
Photophysical property
Azo-hydrazone tautomerism
pKa
Ó 2014 Elsevier B.V. All rights reserved.
Introduction
delocalization through aromatic moieties. Therefore, they have
been used as dyes, known as azo dyes, in textile, food, printing
Azo compounds, bearing the functional group R–N@N–R0, in
and cosmetic industries [1,2]. The rich chemistry of the azo com-
pounds is associated with several important biological reactions
such as protein synthesis, carcinogenesis, azo reduction mono-
amine oxidase inhibition mutagenic, immune chemical affinity
labeling, nitrogen fixation, important medical and industrial uses
[3,4]. Recently, in the last two decades, there has been a rapid
which R and R0 can be either aryl or alkyl, exhibit vivid colors,
especially reds, oranges, and yellows resulted from
p-electron
⇑
Corresponding author. Tel.: +91 381 2346630; fax: +91 381 2346360.
E-mail address: t_k_misra@yahoo.com (T.K. Misra).
http://dx.doi.org/10.1016/j.saa.2014.12.027
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

186
D. Debnath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
growing interest for their potential applications in miscellaneous
areas such as photochromic materials, non-linear optical (NLO)
devices, liquid-crystalline displays (LCDs), dye-sensitized solar
cells (DSSCs), sensors and indicators as well as biological-medicinal
studies [5–10].
6-aminouracil has been reported. The crystal structure of Cu(II),
and Rh(III) complex of 1,3-dimethyl-5-(phenylazo)-6-aminouracil
had been reported [38,37]. A formation of super-complex of the
cationic form of 1,3-dimethyl-5-(phenylazo)-6-aminouracil with
AuXꢁ2 (X = Cl or Br) and its crystal structure had also been reported
[39,40]. In this contribution, we report (i) a modified route of syn-
thesis of four azo-derivatives of 1,3-dimethyl-6-aminouracil
(Scheme 1); (ii) first time the X-ray crystal structure of 1,3-
dimethyl-5-(phenylazo)-6-aminouracil of its azo-tautomeric form
which is found stable in the solid state, (iii) the study of solvato-
chromic property in various organic solvents with different
hydrogen bonding parameters and azo-hydrazone tautomeric
equilibrium in aqueous solution of varying pH to evaluate the
pKa values, and (iv) the potential photophysical property of the
derivatives of 1,3-dimethyl-5-(arylazo)-6-aminouracil.
Among the azo dyes, heterocyclic azo dyes attract considerable
interest and play an important role in the development of the
chemistry of azo compounds. Furthermore, some heterocyclic azo
compounds have also found use as ligands to generate a special
category of metal azo complexes which are exploited enormously
in the manufacture of colorimetric sensors-which lend themselves
to cations and anions [11–13]. Of the heterocycleazo dyes, pyri-
done and pyrimidine derivatives are relatively recent heterocyclic
intermediates for the preparation of aryl-azo dyes [14,15].
Moreover, the applications of azo-dyes are strongly dependent
on the photophysical properties of azo-hydrazone tautomerism
which has been used to some extent for dye location characteriza-
tion in surfactant micelles and textile fibers [16,17], photographic
systems [18], dyeing protein [19,20], bleaching [21,22], and probe
molecules to characterize catalysts [23,24]. Both the tautomers
exhibit different optical and physical properties. The hydrazone
form that absorbs light at longer wavelengths was therefore found
to be rendered higher photoconductivity to dual-layer photorecep-
tors [18] and it is therefore often commercially preferred. The phe-
nomenon of azo-hydrazone tautomerism correlates the mobility of
a labile proton across a molecule through a conjugated system
where the proton remains in association with intra-molecular
hydrogen bonding between donor–acceptor atoms. Such phenom-
enon arises when azo dyes contains OH or NHR group conjugated
with the azo group.
Study of azo-hydrazone tautomerism mechanism is therefore
extremely important and highly demanding for controlling the
molecular properties of these systems depending on the polarity
and pH of the medium. The study of this property on some dyes
has been accomplished and reported [22,25–28] elsewhere. The
above observations have drawn our attention and motivated to
investigate such interesting property of azo-hydrazone tautomer-
ism on azo-dyes with a backbone of 6-aminouracil derivative.
Uracil derivatives, which have pyrimidine-like structure
(uracil = pyrimidine-2,4-dione), have an elegance role in heterocy-
clic chemistry [29]. They have aroused much interest owing to
their potential applications in medicine and photobiology [30].
Pyrimidine dyes in some cases prepared from 6-aminouracils have
found industrial applications in hypnotic drugs [31], in living cells,
in detecting cancer [32] and having pharmacological and biological
activities [33]. The detail synthesis of azo derivatives of uracil has
been paid attention recently and aryl/hetarylazo derivatives of ura-
cil or 1,3-dimethyl-6-aminouracil have been reported [34–37] by
several authors. However, so far no report on the isolation of single
crystal X-ray structure analysis of azo-derivatives of 1,3-dimethyl-
Experimental
Material and instruments
All chemicals were of reagent grade, purchased from Merck,
Aldrich, and Himedia and were used without further purification.
All solvents were of A. R. grade and used for synthesis and spectro-
scopic studies. 1,3-Dimethyl-6-aminouracil was prepared follow-
ing the reported method [41]. For the studies of equilibrium
constant double distilled water was used.
Standard Buffer solutions with pH 1.5, 2.0, 3.48, 3.8, 4.39, 4.76,
5.13, 6.04, 9.0, 9.6, 10.0, 10.4, 10.8, and 12.0 were prepared by fol-
lowing the traditional procedure from double distilled water with
HCl, NaOH, Na2B4O7, NaHCO3, Na2CO3, and KCl. The constant ionic
strength of 0.1 M was maintained with KCl solution. In most cases,
the pH needed to be adjusted using a pH meter and the drop-wise
addition of either 1 M HCl or 1 M NaOH to 1 L of solution. The accu-
rate pH for each buffer solution was measured with a Systonics
digital pH meter 335.
Melting points were determined on a Labtech Digital melting
point apparatus with a heating rate of 2 °C/min and not corrected.
IR spectra were recorded on a Perkin Elmer; model RX-1 FT-IR
spectrophotometer in the region 4000–400 cmꢁ1 making KBr pel-
lets of all dyes. 1H NMR and 13C NMR spectra were recorded on a
Bruker (AC) 300 MHz and a Bruker DRX-500 MHz FT-NMR spec-
trometer in CDCl3 as the solvent and TMS the internal standard.
The electronic spectra were determined on a Shimadzu UV–vis-
1800 spectrophotometer. Quartz cuvettes were used for measure-
ments in solution. Fluorescence spectra of all dyes were recorded
in different solvents on a Perkin Elmer spectrofluorometer, model
LS55. Excitation and emission slits were set to 10 and 5 nm, respec-
tively. Elemental analyses were made by a Perkin Elmer 2400
series-II analyzer and the results agreed well with the calculated
values.
R
NH2
0-5oC
NaNO2/HCl
H3C
H3C
O
H3C
H3C
O
R
N2+Cl-
N
N
N
N
O
N
O
AcOH-H2O/Sirr. at 0-5oC
pH 5-6 with NaOH soln.
N
R
NH2
NH2
R= H (1), CH3 (2), Cl (3), NO2 (4)
Scheme 1. A general reaction scheme of synthesis of dyes (1–4).

D. Debnath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
187
Synthesis of the dyes
Ar–H), 7.36 (s, 3H, Ar–H), 3.80–3.44 (m, 6H, N–CH3); 13C NMR
(125 MHz, CDCl3): d (ppm) 27.16 (CH3), 28.17 (CH3), 114.21 (C),
115.99 (CH), 125.64 (CH), 127.01 (C), 145.22 (C), 163.31(C@O),
165.56 (C@O), 175.35 (C). Anal. Calcd. for C12H12N6O4: C, 47.37;
H, 3.98; N, 27.62, Found: C, 47.29; H, 3.92; N, 27.54.
All the dyes were prepared following the conventional diazoti-
zation process with some modification. As a typical example,
aniline (0.651 g, 7.0 mmol) was precipitated out as a salt of aniline
hydrochloride and made a solution by adding 5 mL of water. It was
then placed in an ice bath and cooled down its temperature to
0–5 °C. An aqueous solution of sodium nitrite (0.484 g, 7.0 mmol
in 10 mL of water) was added slowly into the cooled stirred ani-
line-hydrochloride solution. A light yellow solution, generally
called diazotized solution of aniline, formed and was allowed to
stir further 30 min at the same temperature. 1,3-Dimethyl-6-
aminouracil (1.0 g, 6.46 mmol) was dissolved in hot glacial acetic
acid (5 mL) and to make a clear solution 3 mL of distilled water
was added. The solution was also then placed in an ice bath to cool
it to 0–5 °C. To this solution the diazotized solution was then added
slowly with constant stirring for 30 min. A solution of sodium
hydroxide (4%) was instilled into the reaction mixture to make
the pH ꢂ 5–6. A bright yellow color precipitation was started to
appear. The stirring was continued at r.t. for additional 1 h. The
resulting precipitate was filtered, washed with cold water and
dried over air oven. The product was recrystallized from hot etha-
nol and gave brown plate crystals of 1,3-dimethyl-5-(phenylazo)-
6-aminouracil.
X-ray crystallography
The determination of the crystal class, orientation matrix and
accurate unit cell parameters of the dye, 1,3-dimethyl-5-(pheny-
lazo)-6-aminouracil (0.45 ꢃ 0.38 ꢃ 0.32) was performed using a
Bruker APEX-II CCD diffractometer with graphite monochroma-
tised Mo-Ka (k = 0.71073 Å) radiation. The crystal was kept at
295(2) K during data collection. The structure was solved using
Olex2 structure solution program by following Charge Flipping
solution method [42,43]. All the non-hydrogen atoms were refined
with anisotropic thermal parameters. All hydrogen atoms were
located by geometrical calculation and rode on the parent atom
with isotropic thermal parameters (Uiso(H) = 1.2 Ueq(C or N)).
The structure was refined with the SHELXL-2013 [44] refinement
package using Least Squares minimization. Crystallographic data
and the parameters of structure refinement are summarized in
Table 1.
Determination of pKa values by using spectrophotometry
1,3-dimethyl-5-(phenylazo)-6-aminouracil (1,3-DM-5-PA-6AU) (1)
Bright yellow crystal, Yield: 78%; M.pt. 258–260 °C; FT-IR (KBr,
For the determination of acidity constant (pKa) values of all the
investigated dyes, a stock solution (1.0 ꢃ 10ꢁ3 mol/dm3) of known
weight of a particular dye was prepared in DMSO solvent. Required
volume of dye solution from the stock solution was put into
the aqueous buffer solution to make the final concentration in
the order of 1 ꢃ 10ꢁ5 mol/dm3 in the desired buffer solution
(20:1, v/v aq. buffer/DMSO). After the preparation of a set of
solutions in different buffers, the solution mixtures were settled
for 30 min. UV–vis absorption spectra of all the dyes were recorded
thereafter. The pKa values of all the investigated dyes were
calculated from the obtained spectra following two well-known
methods [45]: (1) half-height method [45a] and (2) modified
t
cmꢁ1). 3274 (–NH2), 1707, 1628 (–C@O), 1523 (–C@C), 1454
(–N@N–), 1357, 1155 (–C–N); 1H NMR (CDCl3, d ppm). 14.12 (br,
1H, –N–NH–), 8.67 (br, 1H, –NH–), 7.72–7.57 (m, 2H, Ar–H), 7.42
(s, 3H, Ar–H), 3.73–3.36 (m, 6H, N–CH3); 13C NMR (125 MHz,
CDCl3): d (ppm) 27.2 (CH3), 27.89 (CH3), 117.27(CH), 127.26 (CH),
129.76 (CH), 144.12 (C), 151.23 (C@O), 153.45 (C@O), 163.33 (C).
Anal. Calcd. for C12H13N5O2: C, 55.59; H, 5.05; N, 27.01, Found: C,
55.47; H, 4.99; N, 26.97.
1,3-dimethyl-5-(p-methylphenylazo)-6-aminouracil (1,3-DM-5-p-
Me-PA-6AU) (2)
Yellow crystal, Yield: 83%; M.pt. 264–262 °C; FT-IR (KBr,
t
cmꢁ1). 3265 (–NH2), 1700, 1622 (–C@O), 1526 (–C@C), 1450
(–N@N–), 1350, 1166 (–C–N); 1H NMR (CDCl3, d ppm). 14.18 (br,
1H, –N–NH–), 8.87 (br, 1H, –NH–), 7.75–7.62 (m, 2H, Ar–H), 7.40
(s, 3H, Ar–H), 3.72–3.34 (m, 6H, N–CH3); 13C NMR (125 MHz,
CDCl3): d (ppm) 21.2 (CH3), 27.24 (CH3), 28.52 (CH3), 116.27(C),
117.26 (CH), 129.76 (CH), 137.59 (C), 138.46 (C), 149.23 (C@O),
151.45 (C@O), 164.33 (C). Anal. Calcd. for C13H15N5O2: C, 57.13;
H, 5.53; N, 25.63, Found: C, 57.01; H, 5.48; N, 25.55.
Table 1
Crystal data of 1,3-dimethyl-5-(phenylazo)-6-aminouracil (1).
Empirical formula
C12H13N5O2
Formula weight
Temperature
Wavelength
Crystal system
Space group
259.27
295.15 K
0.71073 Å
Monoclinic
P21/c
Unit cell dimensions
1,3-dimethyl-5-(p-chlorophenylazo)-6-aminouracil (1,3-DM-5-p-Cl-
PA-6AU) (3)
a (Å)
b (Å)
c (Å)
V (Å3)
Z
10.4315(11)
8.1867(9)
14.5684(15)
1216.5(2)
Yellow crystal, Yield: 72%; M.pt. 262–261 °C; FT-IR (KBr,
t
cmꢁ1). 3289 (–NH2), 1712, 1628 (–C@O), 1523 (–C@C), 1448
(–N@N–), 1352, 1162 (–C–N); 1H NMR (CDCl3, d ppm). 13.87 (br,
1H, –N–NH–), 9.00 (br, 1H, –NH–, 7.83–7.67 (m, 2H, Ar–H), 7.46
(s, 3H, Ar–H), 3.81–3.46 (m, 6H, N–CH3); 13C NMR (125 MHz,
CDCl3): d (ppm) 26.94 (CH3), 28.12 (CH3), 115.97(C), 118.02 (CH),
129.34 (CH), 131.57 (C), 139.14 (C), 158.41(C@O), 165.35(C@O),
172.39 (C). Anal. Calcd. for C12H12ClN5O2: C, 49.07; H, 4.12; N,
23.84, Found: C, 49.02; H, 4.04; N, 23.78.
4
Dcal (Mg mꢁ3
)
1.416
Absorption coefficient (mmꢁ1
F(000)
)
0.102
544
Crystal size
0.45 ꢃ 0.38 ꢃ 0.32
3.994–56.652
ꢁ13 6 h 6 12, ꢁ10 6 k 6 10,
ꢁ19 6 l 6 19
2H range (°)
Index ranges
Reflection observed
3022 [R(int) = 0.0575]
Full-matrix least-squares on F2
3022/0/173
R1 = 0.0466, wR2 = 0.1167
R1 = 0.1013, wR2 = 0.1539
0.859
Refinement method
Data/restraints/parameters
Final R indices [I > 2
1,3-dimethyl-5-(p-nitrophenylazo)-6-aminouracil (1,3-DM-5-p-NO2-
PA-6AU) (4)
r
(I)]
R indices (all data)
Yellow crystal, Yield: 64%; M.pt. 288–286 °C; FT-IR (KBr,
Goodness-of-fit on F2
t
cmꢁ1). 3389, 3256 (–NH2), 1680, 1612 (–C@O), 1511 (–C@C),
Largest difference peak and hole
0.20 and ꢁ0.17
1434 (–N@N–), 1344, 1153 (–C–N); 1H NMR (CDCl3, d ppm).
(e Åꢁ3)
14.49 (br, 1H, –N–NH–), 9.33 (br, 1H, –NH–), 7.63–7.47 (m, 2H,

188
D. Debnath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
limiting absorption method [45b] from the absorption-pH relation-
ship. Moreover, it is the Henderson–Hasselbaltch Eq. (1) which
relates pH and pKa to the equilibrium concentration of anionic
(Aꢁ) and neutral form of an acid (HA).
‘A’ in the solid state. The peaks in the range 1100–1380 cmꢁ1 can
be attributed to the m(C–N) stretching frequency [48]. The weak
bands in the region 943–1058 cmꢁ1 are assigned to be ring vibra-
tions. The moderately sharp strong bands at 759 and 692 cmꢁ1
are due to the stretching vibrations of N–C@O bonds. The out of
pH ¼ pKa þ log½Aꢁꢄ=½HAꢄ
ð1Þ
plane bending,
q(N–H) of N–H bond of the amino group appear
at 483 cmꢁ1 [45]. A representative spectrum (1) is given in Fig. 1.
Thus, the IR data reveal that in the solid phase the azo-dyes exist
in an azo-enamine-keto (A) form.
Results and discussion
The 1H NMR spectral data are also given in the experimental
section and a representative spectrum of the dye 1 is shown in
Fig. 2. The 1H NMR spectrum of 1,3-dimethyl-5-(phenylazo)-6-
aminouracil (1) measured in CDCl3 at 25 °C shows multiplet signals
at d 3.4–3.7 ppm for six N–CH3 protons of the uracil ring. The two
ortho-proton of the aromatic ring appear as multiplet signals at
7.6–7.7 ppm whereas remaining three aromatic ring protons as
singlet signal at 7.4 ppm. The tautomeric hydrazone proton (@N–
NH–) and imine (@NH) protons appear as broad singlet signal at
14.1 ppm and 8.65–8.67 ppm, respectively. There is no signal
found for the –OH proton, ruling out again the presence of the tau-
tomer C (Scheme 2). The results are consistence with the reported
values [49]. The result may be suggested that the tautomeric form,
hydrazone-imine-keto (B) (Scheme 2) was predominantly present
in the solution. Further evidence was obtained from the study of
UV–vis spectra of the dyes in solution.
Synthesis and structure
The azo derivatives (azo-dyes), 1,3-dimethyl-5-(arylazo)-6-
aminouracil (1–4) were prepared by following the modified
diazo-coupling process. The diazotized aniline derivatives were
subjected for coupling with 1,3-dimethyl-6-aminouracil in acetic
acid–water solution followed by neutralization with NaOH solu-
tion to prepare the desired azo-dyes (1–4). The structures of these
dyes were established by studying spectroscopic analysis (UV–vis,
FTIR, 1HNMR and 13C NMR), and X-ray crystal structure. The dyes
are fluorescent active and thereby they were subject for studying
their fluorescent property in solution. The as-synthesized dyes
can exist in three tautomeric forms: azo-enamine-keto (HLazo
(A)), Hydrazone-imine-keto (HLhydrazo (B)), and azo-imine-enol
(HLazo-enol (C)) as shown in Scheme 2.
IR and 1H NMR spectral studies
Crystal structure of 1
The IR and NMR spectral data for all the isolated azo-dyes (1–4)
are given in the experimental section. The
m
(N–Hstrt.) of primary
The molecular structure of the molecule, 1,3-dimethyl-5-
(phenylazo)-6-aminouracil (1) along with the atom numbering
scheme is shown in Fig. 3. The selected bond lengths and bond
angles are given in Table 2. In uncoordinated azo compounds and
hydrazines, the N–N distances are 1.25 [50] and 1.45 Å [51],
respectively. In the present azo-dye (1), the azo, N(4)–N(5) dis-
tance (1.274 Å) is about 0.024 Å longer than the uncoordinated
N–N distance, 1.25 Å. The result is comparable with the results
(azo-N–N, 1.28–1.30 Å) of two reported crystal structures of this
azo-dye where it was found as a protonated form and was isolated
with the counter anion AuClꢁ2 [52] or AuBr2ꢁ [53]. In these two
reported papers [52,53], authors demonstrated that the proton-
ation of an azo-nitrogen by the extra proton resulted in longer
azo bond length. However, our isolated neutral single crystal (1),
it would be quite apparent from the values of other bond distances
(Table 2) that the little longer azo-bond distance may be due to the
association of intra-molecular hydrogen bonding rather than pro-
tonation. The successive bond lengths involving atoms, N(3),
C(7), C(10), N(4), and N(5) are interesting that indicate an existence
aromatic amines generally appear as bifurcated bands at 3450
and 3390 cmꢁ1 [46]. It is somewhat a broad, sharp and strong band
at 3274 cmꢁ1 for the as-synthesized azo dye (1) which is assigned
to the N–H stretching frequency of –NH2 group of the uracil ring
[47,48] at the 6-position. The shifting of the band frequency in
lower region may be suggested that the –NH2 group is associated
in intra- and inter-molecular hydrogen bonding with the azo-
group and keto-group, respectively in the solid state. It has also
been evidenced from the crystal structure of the compound (1).
The aromatic C–H stretching frequency band is overlap with the
N–H (6-amino) signal and appeared in the range 3000–
3200 cmꢁ1
. The sharp intense peaks appeared at 1707 and
1628 cmꢁ1 for the dye 1 are due to the carbonyl groups at 2-
(2C@O) and 4-(4C@O) position of the uracil ring, respectively
[47], which rules out the presence of tautomeric form ‘C’. The
another pair of sharp peaks at 1523 and 1454 cmꢁ1 are assigned
as m(C@C) and m(N@N), respectively. The appearance of the azo-
peak at 1454 cmꢁ1 confirms the existence of the tautomeric form
R
R
R
O
N
N
NH
NH
O
HO
N
N
N
N
5
4
H
6
N
N
N
O
NH
H
N
3
N
N
1
O
2
O
hydrazone-imine-keto
azo-imine-enol
HLazo-enol
azo-enamine-keto
(A)
HLazo
(B)
HLhydrazo
(C)
R = H (1), Me (2), Cl (3), NO2 (4)
Scheme 2. Possible tautomeric forms of 1,3-dimethyl-5-(arylazo)-6-aminouracil: azo-enamine-keto (A), hydrazo-imine-keto (B), azo-imine-enol (C).

D. Debnath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
189
Fig. 1. IR spectrum of the dye 1.
Fig. 2. 1H NMR spectrum of the dye 1.
of six-membered ring current (resonance hybrids) through these
atoms by the association of N(3)–H(3B)ꢅ ꢅ ꢅN(5) [= 1.97 Å) hydrogen
bonding. The Nazo–C (phenyl or uracil) bond distances are also
do not differ much from the expected values. The bond angles of
the phenyl ring are also as expected. The molecule is almost planar
with a dihedral angle about 10.00(7)° between the benzene ring
and the uracil ring.
indicative that the azo-p-electrons are in pull effect toward the
uracil ring. This exo-cyclic C(9)–O(1) [= 1.227(2) Å] bond distance
is slightly longer than that of the other exo-cyclic C(8)–O(2) bond
[= 1.212(2) Å]. It may be the fact that the O(1) atom is associated
with the intermolecular hydrogen bonding with H(3B)–N(3). Thus,
from the results, it evidences that the dye is in azo-enamine-keto
(A) form in the solid state. The bond angles within the uracil ring
Analysis of the crystal packing of the dye in its unit cell shows
the presence of intra- and inter-molecular hydrogen bonding inter-
actions (Fig. 4). The hydrogen bonding parameters are given in
Table 3. The N(3) atom of the 6-aminouracil moiety acts as double
two-center hydrogen bond donors. On the other hand, the one
nitrogen atom of azo-group, N(5) act as single two center hydrogen

190
D. Debnath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
Table 3
Hydrogen bond distances and angles of the crystal structure of 1.
D
H
A
d(Hꢅ ꢅ ꢅA)
2.05
1.97
d(Dꢅ ꢅ ꢅA)
2.844(2)
2.605(2)
<(D–Hꢅ ꢅ ꢅA)
153.0
129.6
N(3)
N(3)
H(3A)
H(3B)
O(1)i
N(5)
Symmetry code: (i) +x, ½ ꢁ y, ꢁ½ + z.
bond acceptor. The dye units, 1,3-dimethyl-5-(phenylazo)-6-
aminouracil, are linked by intermolecular hydrogen bond forma-
tion through N(3)–H(3A)ꢅ ꢅ ꢅO(1), leading a one-dimensional hydro-
gen bonded network along b-axis as shown in Fig. 4. Apart from
that N(3)–H(3B) forms N(3)–H(3B)ꢅ ꢅ ꢅN(5) intra-molecular hydro-
gen bond with the azo-group.
UV–vis absorption and solvatochromism studies
The electronic absorption spectra of the dyes (1–4) were stud-
ied over the wavelength range of 200–800 nm, in organic solvents
of different hydrogen bonding parameters (dH) [54], viz. non-polar:
hexane (Hex) (0.0) and CHCl3 (5.7); polar-aprotic: CH2Cl2 (DCM)
(7.1), DMSO (10.2), and DMF (11.3); polar protic: EtOH (19.4),
and MeOH (22.3). The aspect of UV–vis spectra is either broad or
a peak with a shoulder at longer wavelength as shown in the
Fig. 5 (Fig. 5A (1); Fig. 5B (2); Fig. 5C (3); Fig. 5D (4)). It can be sug-
gested that the dyes were in an equilibrium mixture of tautomeric
forms (A and B, Scheme 2) or protonated and deprotonated forms
of the tautomeric hydrazone form (Scheme 3) in these solvents.
Their UV–vis data are given in Table 4. There are two well-defined
absorption bands at the wavelength range 260–270 nm and 360–
415 nm observed in each spectrum. The former strong band may
Fig. 3. ORTEP view of the dye 1 with atom labeling scheme and 50% probability
thermal ellipsoid.
Table 2
Selected bond lengths (Å) and bond angles (°) of the crystal structure of 1.
Bond length (Å)
O(2)–C(8)
N(3)–C(7)
O(1)–C(9)
N(1)–C(8)
N(1)–C(9)
N(1)–C(12)
N(2)–C(7)
N(2)–C(8)
N(2)–C(11)
N(4)–N(5)
1.212(2)
1.314(2)
1.227(2)
1.374(2)
1.399(3)
1.465(3)
1.370(2)
1.388(3)
1.468(2)
1.274(2)
N(4)–C(10)
N(5)–C(4)
C(1)–C(2)
C(1)–C(6)
C(2)–C(3)
C(3)–C(4)
C(4)–C(5)
C(5)–C(6)
C(7)–C(10)
C(9)–C(10)
1.379(2)
1.427(3)
1.367(3)
1.378(4)
1.383(3)
1.381(3)
1.388(3)
1.377(3)
1.409(2)
1.429(3)
be assigned to the
p ?
p⁄ electronic transitions within the aro-
matic moiety. Sometimes it was difficult to record because of sol-
vent cut-off wavelength in the UV-region. The later absorption
band can be attributed to an overlapping band of n ? p⁄ and
?
p⁄ electronic transitions within the hydrazone/azo form of
Bond angles (°)
C8–N1–C9
C8–N1–C12
C9–N1–C12
C7–N2–C8
C7–N2–C11
C8–N2–C11
N5–N4–C10
N4–N5–C4
C2–C1–C6
C1–C2–C3
C4–C3–C2
C3–C4–N5
C3–C4–C5
C5–C4–N5
p
125.05(17)
116.13(18)
118.80(17)
123.01(16)
120.27(17)
116.72(17)
117.70(16)
114.39(16)
119.9(2)
120.0(2)
120.2(2)
115.39(18)
119.59(19)
125.0(2)
C6–C5–C4
C5–C6–C1
N3–C7–N2
N3–C7–C10
N2–C7–C10
O2–C8–N1
O2–C8–N2
N1–C8–N2
O1–C9–N1
O1–C9–C10
N1–C9–C10
N4–C10–C7
N4–C10–C9
C7–C10–C9
119.5(2)
120.7(2)
the dyes [55]. The strong
p ?
p⁄ transition is generally override
the feeble n ? p⁄ transitions which may or may not be identified
from the spectra. The hypsochromic shift of kmax in each case of
as-synthesized dyes (1–4) was observed with the increase of the
value of hydrogen bonding parameters (dH). There was observed
some short of deviation in the chlorinated solvents: CHCl3 and
DCM which may be the some sort of interactions occurred between
the solvents chlorine atom and the solute dyes. Moreover, the
observed kmax shifting (hypsochromic) is more concomitant to
hydrogen bonding parameter than to the dipole moment parame-
ter (bathochromic) of the various solvents [1,56]. In the present
study, the effect of polar aprotic and polar protic solvents on the
118.97(17)
121.44(18)
119.59(17)
122.0(2)
121.66(19)
116.32(17)
118.50(19)
125.26(19)
116.23(16)
126.05(18)
114.28(16)
119.67(17)
Fig. 4. 1D hydrogen bonding network of the dye 1 along the axis b.

D. Debnath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
191
Fig. 5. Solvent effects on electronic absorption spectra of the dyes: 1 (A), 2 (B), 3 (C), and 4 (D).
R
R
O
N
N
NH
NH
O
N
N
N
-
/
OH
Polar Solvents
NH
N
N
O
O
L-hydrazo (B/)
HLhydrazo (B)
Scheme 3. Equilibrium between protonated hydrazone-tautomer, HLhydrazo (B) and deprotonated hydrazone-anion, Lꢁhydrazo (B/) forms.
kmax can also be correlated with the dipole moment parameter; the
red-shifting of kmax occurs with the increase of their dipole
moment parameter. The phenomenon is quite different in case of
non-polar solvents. Thus to study the solvent effects considering
of hydrogen bonding parameter of the solvents are quite signifi-
cant. The observed phenomenon is also given a strong indication
in the presence of protonated and deprotonated equilibrium rather
than azo-hydrazone equilibrium in solution. The solvents having
greater hydrogen bonding parameter could stabilize the deproto-
nated form of hydrazone more effectively than the protonated
form. Consequently, blue-shifting of kmax occurs with the increase
of dH of the solvents. It can be attributed to the stabilization of the
ground state of the deprotonated form through hydrogen bonding
interactions. The fact is quite true for the dyes 1–3. Nevertheless,
the presence of nitro-substituent at the para-position of the ben-
zene ring of the dye 4 destabilize the ground state by bring up
the negative charge onto its head through resonance, as shown
in Scheme 4, resulting the reduction of band gap. The kmax of the
dye 4 in all solvents is therefore appeared in the red-region com-
pared to the other dyes (1–3).
In study of tautomeric equilibrium in acid-base media (later
section), the spectrum of each dye exhibited a certain isobestic
point which may also be suggested the existence of an equilibrium
between hydrazone and hydrazone/azo-anion. The absorption
spectra of dyes in hexane and chloroform solvents show single
broad absorption bands which have found at the red-region from
the far way of their isobestic points. In these two solvents the pre-
dominant form of the dyes may be the hydrazone form (B)
(Scheme 2). The result is also concomitant with the 1H NMR study.
In other solvents the dyes exhibit a strong absorption band with a
shoulder at longer wavelength, indicating the separation of n ? p⁄
transition from the
p ? . The absorption band at shorter
p⁄

192
D. Debnath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
Table 4
Study of fluorescence property and solvent effects
Influence of solvent on the electronic absorption properties of azo-dyes (1–4)
(C = 1 ꢃ 10ꢁ5 mol/dm3).
Compounds associated with lowest-energy
p ?
p⁄ transition
Solvent (dH, H-bonding
parameter)
kmax (nm)
exhibit most intense and most useful fluorescence property rather
than n ? p⁄ transition [58]. The lowest-energy electronic transi-
tion of our as-synthesized dyes is ascribed as an overlapping strong
1
2
3
4
Hex (0.0)
CHCl3 (5.7)
DCM (7.1)
387
390
394
399
383
386
396
401
400
p
?
p⁄ electronic transition along with weak n ? p⁄ transition of
369,
404sh
368,
408sh
366,
406sh
361,
402sh
360,
401sh
400
373,
396sh
372,
412sh
370,
410sh
364,
410sh
363,
410sh
411
377,
406sh
374,
414sh
372,
416sh
370,
407sh
368,
409sh
400
the hydrazone form. The UV–vis study in different solvents as well
as in acid-base media of all the dyes (1–4) shows this band in the
region of 412–360 nm. Thus the fluorescent property of the dyes
was investigated in different solvents. Upon excitation at particular
wavelength the dyes (e.g. for 1, kex = 368 nm in DMSO and
kex = 366 nm in DMF solvents) exhibit strong fluorescent peak (1,
kem = 437 nm for DMSO; kem = 431 nm for DMF). The spectra of
all the dyes (1–4) showing emissions in different solvents are por-
trayed in the Fig. 6 (Fig. 6A (1); Fig. 6B (2); Fig. 6C (3); Fig. 6D (4))
and the corresponding data are given in the Table 5. The kem values
obtained from fluorescence spectra are comparable with the
obtained electronic absorption data in respect of behavior. How-
ever, the most noticeable point is the observed intensity difference
of the dyes especially for the dyes 1–3 at their corresponding kem in
different solvents. The observed intensity is in high value in each
case in DMSO and DMF whereas in other solvents it is quite low.
This observation may be suggested that the fluorescence property
of these dyes is due to their anionic forms which are in predomi-
nant form in these solvents. Zhang et al. [59] demonstrated that
the anionic form of benzotriazole exhibited more strong fluores-
cence peak than that of its protonated form. DMF and DMSO sol-
vents are the proton acceptor solvents and the existence of the
anionic form bearing negative charge is thereby reasonable in
these two solvents. In contrast, MeOH and EtOH are proton donor
solvents where the negative charge on the anionic form must be in
association with hydrogen bonds with these solvent molecules;
with the result it is expected to be less fluorescent, i.e. low intense
DMSO (10.2)
DMF (11.3)
EtOH (19.4)
MeOH (22.3)
439sh
502
430,
517sh
397
,
397
MeOH + HCl
MeOH + NaOH
401
462
372,
370,
371
418sh
418sh
wavelength of each dye is ascribed as
p ?
p⁄ transition. Except in
DCM, thus, there is a clear trend of blue-shifting of shorter wave-
length band maximum with the increase of hydrogen-bonding
parameter of solvents from DMSO (11.3) to MeOH (22.3). The
existed equilibrium, in these solvents, may be in between the
forms of hydrazone and its anionic form (Scheme 3). The solvent
having greater value of hydrogen bonding parameter thus assists
deprotonation of the hydrazone form by stabilizing the deproto-
nated anionic form, with the result that the shorter wavelength
band is blue-shifted (hypsochromic shift) accordingly. It is there-
fore demonstrated that the solvent having high value of dH (3:
Hex (0.0), kmax = 384 nm–MeOH (22.3), kmax = 368 nm) could solvate
or stabilize the anionic form of dyes more effectively than the
solvent of low dH. The solvatochromic behavior of the as-synthesized
azo-dyes is thereby tagged as positive solvatochromism.
To study the effects of acid and base on the absorption maxima
of the dyes in solvents like MeOH, 1–2 drop of concentrated HCl or
4 M NaOH solution was added into the MeOH solution of all dyes
graph. It may also be suggested that the p ?
p⁄ electronic transi-
tions of the dyes in these two solvents are no longer remained a
lowest-energy transition instead the weaker n ? p⁄ became the
lowest one as it appears as a shoulder peak found in the UV–vis
spectra. In other solvents: Hex, DCM, and CHCl3 the predominant
form is the hydrazone form and is suggested to be less fluorescent
in these solvents as well. The dye 4 exhibits very low intense fluo-
rescent curve which is justified because of the displacement of the
negative charge from the hydrazone head to the nitro-group as
shown in the Scheme 4. The results are therefore again evidenced
that the predominant form of the dyes in polar aprotic and protic
solvents is the anionic form of hydrazone tautomer.
and noted the change of kmax. A bathochromic shift (Dkmax = 40 nm)
was observed in each case in presence of acid and practically no
shift occurred in presence of base for the dyes. The results show
that the dyes could exist in dissociated form i.e. azo- or hydrazone
anion in MeOH which is already demonstrated above and the
added base had nothing to do on the species present in the
solution. The added acid in MeOH, on the other hand, then had
protonated the anionic species present in MeOH solution, resulting
in the transformation from the anionic species to its protonated
hydrazone-tautomeric form (B). Fluorescence results have also
yielded the same result. The data in Table 4 are borne out such
demonstration. The result seems to be compatible with the hydra-
zone form rather than the azo form and is consistent with the 1H
NMR data. The existence of anionic form of hydrazone in case of
non-N-methylated phenylazo-6-aminouracil was also observed in
DMSO and demonstrated by Zeynel and Nermin [56,57].
Electronic absorption spectra: study of tautomeric and acid-base
equilibria
In addition to the azo-hydrazone equilibrium, the dyes can
co-exist in acid-base equilibrium, depending on the pH of the
media [60]. The possible acid-base equilibrium of azo-hydrazone
O-
N
NO2
O
O
O
N
N
N
N
N
N
O
O
N
NH
N
NH
L-hydrazo (B//)
L-hydrazo (B/)
Scheme 4. Canonical forms of the anionic form of hydrazone tautomer of 1,3-Dimethyl-5-(p-nitrophenylazo)-6-aminouracil (4) where Lꢁhydrazo (B/)-negative charge on the azo-
nitrogen and Lꢁhydrazo (B//)-negative charge on the nitro-group.

D. Debnath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
193
Fig. 6. Fluoroscence spectra of the dyes: 1 (A), 2 (B), 3 (C), and 4 (D) in different solvents.
R
Table 5
R
Influence of solvent on the emission properties of azo-dyes (1–4), kem, nm.
Solvent kex, kem/nm (I/a.u.)
O
N
N
NH
NH
1
2
3
4
O
4
N
N
5
kex
kem
kex
kem
kex
kem
kex
kem
H
6
N
N
Hex
387 433(17)
440(15)
390 437(53)
463(36)
369 434(5)
459(3)
368 437(97)
414(74)
394 443(11)
383 429(22)
408(19)
386 438(41)
461(32)
396 439(15)
446(12)
401 437(55)
456(38)
400 549(4)
543(4)
439 498(5)
532(5)
3
N
H
N
1
O
2
O
CHCl3
DCM
DMSO
DMF
399 438(23)
466(20)
373 467(2)
HLhydrazo (B)
HLazo (A)
377 450(5)
372 441(77)
417(70)
370 433(54)
415(41)
374 438(72)
415(61)
372 432(67)
424(63)
H+
Ka(hydrazo)
H+
Ka(azo)
R
R
366 431(89)
412(84)
430 474(6)
504(5)
EtOH
MeOH
361 404(12)
428(7)
360 407(7)
438(7)
364 437(4)
455(3)
363 436(8)
409(6)
370 441(6)
436(5)
368 436(11)
432(9)
397 486(5)
497(5)
397 452(3)
462(3)
O
4
N
N
O
N
N
N
5
6
N
NH
NH
3
N
1
2
N
O
O
L-azo (D)
L-hydrazo (E)
tautomerism of the dyes under investigation is portrayed in
Scheme 5. The tautomeric form hydrazone in the equilibrium,
forms by the transfer of hydrogen atom from the –NH2 group to
the azo-N atom which is susceptible to form hydrogen bonding
with it. Thus, the shifting of a double bond along with a proton
transfer leads to the establishment of such tautomerism. In basic
media, the formation of azo anion (Lꢁazo (D)) and hydrazone anion
(LꢁHydrazo (E)) may be suggested to form by the deprotonation from
the –NH2 group of the azo-form (A) and the –NH group of the hydra-
zone form (B), respectively. These anionic forms are the indistin-
guishable resonance hybrids of azo-anion (D) and hydrazone-anion
(E) canonical forms.
Scheme 5. Possible acid-base and azo-hydrazone equilibria. The different forms are
azo-enamine-keto, HLazo (A), hydrazone-imine-keto, HLhydrazo (B), azo-anion, Laꢁzo
(D), hydrazo-anion, Lꢁhydrazo (E), acid dissociation constant for the azo-tautomer
(Ka(azo)), and acid dissociation constant for the hydrazone-tautomer (Ka(hydrazo)).
To study the electronic absorption spectra of these presumed
tautomeric and deprotonated forms, the UV–vis absorption spectra
of the dyes (1–4) were recorded in the pH range 2.0–10.8 in water
and are shown in Fig. 7 (Fig. 7A (1); Fig. 7B (2); Fig. 7C (3); Fig. 7D
(4)). The Table 6 is borne out the corresponding kmax data. The

194
D. Debnath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
Table 6
main absorption band which is assigned as an overlapping band of
n ? p⁄ and p⁄ electronic transitions within the hydrazone
Absorption maxima (kmax) of the dyes 1–4 at different pH of buffer solutions.
p
?
form [55], is affected most by the change of pH of the media. More-
over, the spectral change (dye 1) from the pH 2.0 (kmax = 401 nm)
to pH 10.8 (kmax = 360 nm) is noticeable with a well-defined iso-
bestic point at 371 nm. Analogous phenomenon is also observed
for the dye 2 as well as 3. The isobestic point means the existence
of an equilibrium between the deprotonated (anionic form: Lꢁazo or
Lꢁhydrazo) and protonated (HLhydrazo) form of hydrazone. In high acidic
media (pH 2.0–3.48), the kmax of the dyes is affected negligibly, indi-
cating that the hydrozone is the predominant form. As the pH value
increases, the intensity of the band decreases initially and further
increase of pH leads to the hypsochromic shifts of the kmax which
passes through an isobestic point. In basic media, the absorption
pH
kmax (nm)
1
2
3
4
2.0
3.48
3.80
4.39
4.76
5.13
6.04
9.0
9.6
10.0
10.4
10.8
402
401
399
400
413
411
411
411
401
399
399
395
404
404
403
402
401
400
367sh, 393
362, 404sh
360, 406sh
360, 406sh
360, 406sh
360, 406sh
361, 406sh
360, 406sh
371
367sh, 405
367, 404sh
364, 406sh
363, 406sh
362, 406sh
362, 406sh
362, 406sh
362, 406sh
380
387
372, 398sh
369, 406sh
364, 408sh
362, 410sh
362, 410sh
362, 412sh
362, 412sh
374
399, 454sh
400, 454sh
401, 454sh
404, 454sh
408, 454sh
405, 454sh
band, assigned as
p ?
p⁄ transition, is suggested to the absorption
Isobestic point
of the common resonance anion which is the conjugate base of azo
and hydrazone tautomers (Scheme 5). Besides, a low intensity shoul-
der peak appeared at ꢂ400 nm in basic medium is assigned as
n ? p⁄ transition which is less sensitive to pH changes to push–pull
effects. Unlike dyes 1–3, for the dye 4 the intensity of the peak at
kmax decreases and becomes broaden without shifting much anyway
with the increase of pH. Apart from a shoulder type of peak develop-
ment occurs in the red region. The results reveal the decrease of con-
centration of the anionic form of hydrazone (Lꢁ(hydrazo) (B/)) and the
development of the new species Lꢁ(hydrazo) (B//) due to the strong –I
effect of the –NO2 group as shown in Scheme 4.
Sh: shoulder.
buffer solutions of varying pH 2.0–10.8 at 25 2 °C and are given
in Table 7. The concentration of the dyes (1–4) in the buffer solu-
tion of pH 2.0–10.8 was maintained at 1 ꢃ 10ꢁ5 mol/dm3. The ionic
strength,
l = 0.1 of all the solution was maintained with KCl solu-
tion. The dyes (1–3, Fig. 7A–C) exhibit two bands with kmax in
region of 360–372 for anionic (Lꢁazo or Lhꢁydrazo) and 390–415 nm
for molecular species, hydrazone (HLhydrazo) whereas the dye 4
(Fig. 7D) exhibit a single band at ꢂ404 nm for both the species. As
the pH value of solution increases, the height of the former band
for the dyes (1–3, Fig. 7A–C) increases simultaneously that of the
later decreases; for the dye 4 (Fig. 7D) the band intensity decreases
along with a development of a shoulder peak at longer wavelength,
as shown in Fig. 7. Each dye except 4 exhibits an isobestic point
Determination of pKa values of the azo dyes
The acid dissociation constants (pKa) of the dyes under investi-
gation were determined from their UV–vis spectral behavior in
Fig. 7. UV–vis spectra of the dyes: 1 (A), 2 (B), 3 (C), and 4 (D) in the pH range 2–10.8.

D. Debnath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
195
Table 7
UV–vis spectral data of hydrazone-enamine-keto tautomer of dyes 1–4 and their acid dissociation constants (pKa).
Dye
kmax (HLhydrazo), nm
kmax (Lꢁhydrazo), nm
Half height method
Modified limiting method
pKa (mean)
pKa at kHL
pKa at kꢁL
1,3-DM-5-PA-6AU (1)
401
413
401
404
361
362
362
4.72
5.20
4.70
3.98
4.78
5.08
4.60
5.28
5.15
4.66
4.29
4.92
5.14
4.65
4.12
1,3-DM-5-p-Me-AA-6-AU (2)
1,3-DM-5-p-Cl-AA-6AU (3)
1,3-DM-5-p-NO2AA-6-AU (4)
Fig. 8. Plot of absorbance vs pH: 1 (A), 2 (B), 3 (C), and 4 (D).
pKa(azo) to the Eq. (5) and considering the large value of KT the Eq.
(5) reduces to Eq. (6). The
which is in the range 371–380 nm, indicating the establishment of
equilibrium between the neutral and ionic form. The simplified
acid-base and azo-hydrazone equilibrium is shown in Scheme 5.
The equilibrium constants can be expressed according to the Eqs.
(2)–(4). Where Ka(azo) and Ka(hydrazo) describe the
pKa ¼ pKaðhydrazoÞ
ð6Þ
obtained pKa correspond to pH when the concentration of hydra-
zone and its anionic form is in equal measured. The pKa values were
determined by making use of two spectrophotometric methods,
namely half-height [45a] and the modified limiting absorption
[45b] as depicted in Figs. 8 and 9, respectively and both the meth-
ods gave concordant results. The constructed absorbance-pH rela-
tions at the selected wavelength are sigmoidal curves shaped
(Fig. 8A for 1; Fig. 8B for 2; Fig. 8C for 3; Fig. 8D for 4), each compris-
ing a clear inflection, indicating typical dissociation processes
(Fig. 8). When plotting the log absorbance ratio vs pH, a straight line
is obtained in each case (Fig. 9A for 1; Fig. 9B for 2; Fig. 9C for 3;
Fig. 9D for 4); the zero value of the log absorbance ratio indicating
corresponding pKa value (Fig. 9). The pKa values of all the dyes were
evaluated at their corresponding wavelength and these are listed in
the Table 7. The values of pKa fall in the acid region which may be
suggested that the dissociable proton must be the hydrazone pro-
ton. The determined acid dissociation constants (pKa) values
(Table 7) increases according to the following sequence:
2 > 1 > 3 > 4. The lower pKa value of the dye 4 is justified and this
is due to the presence of the electron withdrawing group at the
para-position of the phenyl ring. In contrast, the dye 2 having + R
effect of the –Me group at the para-position of the phenyl ring pos-
sesses comparably higher value.
½Lꢁazoꢄ½Hþꢄ
KaðazoÞ
¼
ð2Þ
ð3Þ
ð4Þ
½HLazo
ꢄ
½Lꢁazoꢄ½Hþꢄ
KaðhydrazoÞ
¼
½HLhydrazo
ꢄ
½HLhydrazo
ꢄ
KT ¼
½HLazo
ꢄ
acid-dissociation constant of compounds containing azo-enamine
and hydrazone-imine form, respectively and KT is the tautomeric
equilibrium constant. If both the forms, HLazo and HLhydrazo present
in the solution quantitatively, the equilibrium constant can be
describe with macroscopic pKa value as expressed in Eq. (5). Where
pKa(azo) = ꢁlog Ka(azo) and pKa(hydrazo) = ꢁlog Ka(hydrazo). The
1
KT
ð1 þ KTÞ
pKa ¼
ꢅ pKaðazoÞ þ
ꢅ pKaðhydrazoÞ
ð5Þ
ð1 þ KTÞ
study of solution spectra as well as of 1H NMR spectra reveals that
in acidic and neutral solution the most predominant form is the
hydrazone-imine form, thereby neglecting the contribution of

196
D. Debnath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 185–197
Fig. 9. plot of pH vs ꢁlog [A ꢁ Ab]/(Aa ꢁ A) where A = absorbance, a = acid and b = basic.
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The isolation and characterization of a single crystal structure of
an azo dye 1 of 1–4 of the dyes derived from 1,3-dimethyl-6-
aminouracil has been executed. The existence of the tautomeric
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The dyes exhibit prominent photophysical properties in certain
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Acknowledgement
The authors are very grateful to the Department of Chemistry,
NIT, Agartala for providing all kind of supports from all corners.
Of the authors D. Debnath is indeed thankful to NIT, Agartala for
providing him institutional fellowship. The authors are indebted
to Professor C. Sinha for helping in collecting FTIR and 1H NMR
spectroscopic data.
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