Structure investigation of intramolecular hydrogen bond in some substituted salicylaldehydes and 4-aminoantipyrine derivatives in solution and in the solid state

Article abstract of DOI:10.1016/j.saa.2013.01.094

Seven imine derivatives obtained by condensation of appropriate aldehydes and salicylaldehydes with 4-aminoantipyrine were investigated in terms of intramolecular hydrogen bond structure. On the base of ¹H, ¹³C and ¹⁵N NMR

Full text of DOI:10.1016/j.saa.2013.01.094

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 109 (2013) 47–54
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Structure investigation of intramolecular hydrogen bond in some substituted
salicylaldehydes and 4-aminoantipyrine derivatives in solution and in the solid state
Wojciech Schilf a, , Bohdan Kamienski a,b, Anna Szady-Chełmieniecka c, Beata Kołodziej c,
⇑
´
c
d
d
d
´
Eugeniusz Grech , Dorota Zarzeczanska , Anna Wcisło , Tadeusz Ossowski
a Institute of Organic Chemistry, Polish Academy of Sciences, 44/52 Kasprzaka str., 01-224 Warsaw 42, POB 58, Poland
b Institute of Physical Chemistry, Polish Academy of Sciences, 44/52 Kasprzaka str., 01-224 Warsaw 42, POB 58, Poland
c Department of Inorganic and Analytical Chemistry, West Pomeranian University of Technology, Piastów 42, 71-065 Szczecin, Poland
d Faculty of Chemistry, University of Gdan´sk, Sobieskiego18/19 str., 80-952 Gdan´sk, Poland
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
"
We synthesized seven o-hydroxy
Schiff bases from 4-aminoantipyrine.
We performed combined solution
1H, 13C and 15N and solid state 13C
and 15N NMR spectra.
The positions of tautomeric
equilibria of Schiff bases were
defined.
O
O
H
N
CH3
H
N
CH3
"
N
N
X
CH3
N
X
CH3
N
O
O
"
"
The pKa measurements were done to
compare with NMR data.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 October 2012
Received in revised form 23 January 2013
Accepted 28 January 2013
Available online 20 February 2013
Seven imine derivatives obtained by condensation of appropriate aldehydes and salicylaldehydes with 4-
aminoantipyrine were investigated in terms of intramolecular hydrogen bond structure. On the base of
1H, 13C and 15N NMR measurements in solution and in the solid state we found out that all compounds
which can form such structure exist as OH forms with strong H-bonds to nitrogen atom. The structure
conclusions taken from NMR study were confirmed by pKa measurements. Surpassingly, the positions
of protons in H-bridges only very slightly depend on the substituents in aldehyde used for condensation
and on the phase (solution vs. solid state). The influence of antipyrine moiety seems to be the major factor
defining H-bond structure.
Keywords:
H-bond
Schiff bases
Tautomerism
1H
Ó 2013 Elsevier B.V. All rights reserved.
13C
15N
Introduction
material technology (nonlinear optical materials) [3,8,9]. The Schiff
bases were also used in pharmaceutical industry as the compo-
The imines called as the Schiff bases obtained by condensation
of aldehydes and primary amines are well known since 1864 [1,2].
The unique chemical properties of those compounds are the
main reason of very extensive application of Schiff bases in various
areas of organic chemistry: transition metal complexes applied in
homogenous catalysis [3–6], coordination chemistry [7], modern
nents of antibiotics, antibiotics, antiallergic, antiphlogistic and
antitumor substances [2,10–12]. The imino moiety is very useful
active center of many biological systems [13,14]. As a very good
example of this application are extensive study of gossypol-Schiff
bases derivatives studied by Przybylski et al. applying many spec-
troscopic, theoretical and X-ray methods [15,16].
The Schiff bases obtained from different substituted salicylalde-
hydes can form intra- and intermolecular hydrogen bonds, which
mostly determine the chemical and physicochemical properties
of those compounds [17–25]. Various studies have shown that
⇑
Corresponding author. Tel.: +48 223433318; fax: +48 226326681.
E-mail address: wojciech.schilf@icho.edu.pl (W. Schilf).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.01.094

48
W. Schilf et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 109 (2013) 47–54
Schiff bases derived from salicylaldehyde and its derivatives have
considerable biological importance because of presence of many
active donor atoms (N and O) in molecules of these compounds
and being to some extent analogous to biological systems [26].
Imines obtained from heteroaromatic compounds containing
nitrogen atoms in the ring have efficient biological activities [27].
For this reason Schiff bases derived pyrazolones are now studying
as a new kind of chemoterapeutic [28].
Schiff bases obtained from 4-aminoantipyrine (4AAP) exhibit
great variety of biological activities such as antitumor, fungicidal,
bactericidal, antiviral, anti-inflammatory [29], antipyretic, analge-
sic, antiproliferative and antioxidant activities [30–32].
To perform structural studies of Schiff bases many analytical
methods including all spectroscopic measurements and theoretical
calculations were applied. Some of them were presented and re-
viewed in following papers [33–46].
BB probehead at room temperature. Typical CPMAS experimental
conditions for 15N measurements were: spectral width 25 kHz,
acquisition time 30 ms, contact time 4 ms, rotation rate 6–
10 kHz, relaxation delay up to 180 s depending on relaxation prop-
erties of the sample, number of scans about 300. For carbon CPMAS
experiment the following conditions were applied: spectral width
31.250 kHz, acquisition time 30 ms, contact time 2 ms, rotation
rate 10–11 kHz, relaxation delay up to 180 s and about 100 of scans
for each spectrum were collected. For short contact time CH spec-
tra the contact time 40 ls was applied. The basic acquisition
parameters for CPMAS spectra (contact times, acquisition times,
power ratio on both channels during spin-look and relaxation de-
lays), for both carbon and nitrogen measurements, have been opti-
mized to obtain the highest possible signal to noise ratio.
Additionally the spinning rate was individually adjusted to move
the rotational side bands in the region without real signals.
For the present investigations we have chosen five 2-hydroxy
aldehyde derivatives and one 2-hydroxy dialdehyde derivative
(2,6-diformyl-4-methylphenol) capable to form intramolecular
hydrogen bonds, and one model compound – isophtalaldehyde –
which is not able to do this.
UV VIS spectrophotometric measurements
The measurements were carried out by means of spectrophoto-
metric titration. Perkin–Elmer Lambda 650 UV–Vis double beam
spectrophotometer, with automatic stirrer, was used for absor-
bance measurements. All UV–Vis spectra were recorded in 220–
500 nm range, 1.00 cm quartz microcells were used. The com-
pounds were dissolved in acetonitrile:methanol (90%:10%) mixture
and titrated with methanosulfonic acid solution. The same solution
was titrated with sodium hydroxide in acetonitrile:methanol
(90%:10%) mixture. For each point of a known pH the absorption
spectrum was recorded. pH measurements were made with Cerco-
Lab system with a pH-meter using a combined glass electrode. The
electrode was calibrated in the buffer system of 2,6-dinitrophenol/
tetrabutylammonium 2,6-dinitrophenolate in acetonitrile. Concen-
trations of compounds used for all spectrophotometric measure-
ments were around 2 ꢁ 10ꢀ5–5 ꢁ 10ꢀ5 M. All measurements
were performed at 298 K temperature.
To obtain the values of dissociation constants of measured com-
pounds (pKa) the resulting profile of absorbance at the wavelength
of maximum absorption vs pH in each series were used to obtain
the equilibrium constants of complex species tested formed in
each system. All calculations were performed in OriginLab soft-
ware using the Henderson–Hasselbach equation, based on change
in absorption as a function of pH of the solution.
It is known, that 15N NMR is a very good analytical technique to
determining proton localization in hydrogen bond formed between
nitrogen atom of imine group and oxygen atom from phenolic
group. Signals in the spectral range from about ꢀ50 to ꢀ220 ppm
are assigned to the imine groups in different stages of proton trans-
fer process [47,48].
In our previous studies we found out that three factors influ-
ence hydrogen bond structure (proton position in the bridge): (i)
substitution in aromatic ring (the major one), (ii) the amine used
for the imine formation and (iii) the phase (solution or solid state)
[47].
In this paper, we report the synthesis of Schiff bases obtained
from 4AAP and some o-hydroxyaldehydes as well as isophtalalde-
hyde as the model compound for the structure without hydrogen
bond, and structural study using 1H, 13C, 15N NMR in solution
and 13C, 15N CPMAS NMR. To confirm the results obtained by spec-
troscopic method the pKa measurements in basic and acidic condi-
tions were performer.
Experimental
Synthesis
½Bꢂ
Ak ꢀ AkBH
pKa ¼ pH ꢀ log
¼ pH ꢀ log
ꢀ
½BHꢂ
AkB ꢀ Ak
The imine derivatives of 4-aminoantipyrine (4AAP) have been
obtained by condensation in methanol solution of parent amine
and appropriate aldehyde in proportions 1:1. This is the modifica-
tion of the method published previously [49]. The mixture was
stirred and refluxed for 1 h. Then the mixture was cooled to room
temperature. After 24 h the obtained precipitate was filtered out
and washed by methanol. The authenticity and purity of obtained
compounds were examined by proton NMR measurements.
where B is compound in base form and BH is compound in acid
form, AkB is absorbance at the base form and AkBH is the absorbance
at the compound in acid form.
To define the number of equilibria present in the studied sys-
tem, we analyzed the A-diagrams, which show a relationship be-
tween absorbance at two, different wavelengths. The theoretical
model was fitted to experimental data presented as a relationship
of absorbance vs. pH. For the evaluation of electrode parameters
the STOICHIO version CVEQUID software based on the nonlinear
least-squares Gauss–Newton–Marquardt algorithm was used
[50–52]. The resolution of the voltage measurement was <0.1 mV.
NMR measurements
All NMR spectra in solution have been run on Bruker DRX
Avance 500 and Varian VNMRS 600 spectrometers using 5 mm
TBI Z-gradient and auto XID probeheads, respectively at room tem-
perature. The signals assignment has been done on the base of
gCOSY, gHSQC and gHMBC experiments using standard Bruker
and Varian procedures for both acquisition and data processing.
The chemical shifts of proton and carbon signals are referred to
internal TMS whereas the nitrogen-15 chemical shifts are referred
to external nitromethane as a standard. The CPMAS spectra were
done using Bruker Avance II 500 equipped with 4 mm MAS 1H/
Results and discussion
Results of all NMR measurements of all antipyrine derivatives
(Fig. 1) in both solution and solid state are collected in Tables 1
and 2. The liquid state spectra were run on CDCl3 solutions except
2H5MITFAB sample, where due to solubility problem DMSO was
used. Additionally, some experiments in CD3CN solution were per-
formed to look for possible solvent effects. The proton and carbon

W. Schilf et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 109 (2013) 47–54
49
chemical shifts were assigned by analysis of 1D spectra and homo-
and heteronuclear 2D experiments (gCOSY, gHSQC and gHMBC).
The assignment of nitrogen NMR signals was done on the base of
heteronuclear 2D experiments (gHMBC) and analysis of chemical
shifts values characteristic for nitrogen sites present in investi-
gated molecules. The most deshielded signals, close to ꢀ100 ppm
were assigned to imine nitrogen atoms. This assignment was
confirmed by correlation with protons bonded to C-7 atom. The
most shielded signals were assigned to cyclic amine sites. Since
both of those signals are correlated with both methyl groups in
five-membered ring, the differentiation of them can be done by
correlation with aromatic protons. Only signals located close to
ꢀ200 ppm showed such correlation so they must be assign to
N-20 position. In the carbon and nitrogen solid-state spectra the
signals assignment was done by comparison with solution spectra.
Additionally, to distinguish protonated and quaternary carbon
atoms the short-contact time experiments were run.
The most interesting problem in structure analysis of investi-
gated compounds is the structure of intramolecular hydrogen
bonds formed by OH groups with imine moiety. The best spectral
parameter to define position of proton in hydrogen bridge is nitro-
gen chemical shift of imine atom. The general rule is: the most upf-
iels shift of this signal means the strongest proton transfer from
oxygen atom to nitrogen site [53–59]. For aliphatic derivatives
without H-bonds this chemical shift is about ꢀ60 ppm, relative
weak H-bond can shift this signal to about ꢀ100 ppm, for com-
pounds with strong proton transfer nitrogen signal can be shifted
to about ꢀ240 ppm. Position of this signal and consequently posi-
tion of the proton in the bridge are determined by two structural
factors: substituents present in phenyl ring and character of base
used for condensation. Generally, all substituents, which increase
acidity of OH group, promote proton transfer from oxygen to nitro-
gen atom. The influence on amine residue on proton position is
generally smaller and aliphatic derivatives promote stronger pro-
ton transfer comparing with aromatic analogues. The second
parameter suitable for structure elucidation is carbon chemical
shift of formally C-OH atom. In this case we observe opposite ef-
fect, strong proton transfer to nitrogen atom causes the downfield
shift of this signal. This effect is much weaker than those observed
for nitrogen chemical shift. For compounds without H-bonding the
C-2 signal is located close to 155 ppm, proton transfer process can
shift it to about 175 ppm. In contrast to imine nitrogen chemical
shifts the C-2 chemical shifts are less diagnostic since they are also
affected by substituents present in phenyl ring.
The analysis of spectral data should be started from nitrogen
chemical shifts. The first observation is the very narrow range of
changes for imine chemical shifts. For simple aliphatic amine
derivatives this parameter spans much broader range. For example,
for 5-Cl derivative dNs = ꢀ90.7 ppm, for 5-NO2 dNs = ꢀ110 ppm, for
3-MeO, 5-NO2 derivative dNs = ꢀ176.1 ppm and for naphtyl deriva-
tive dNs = ꢀ211.6 ppm [57,58]. It means that for antipyrine deriva-
tives the position of proton in H-bridge is less sensitive on
substituent in phenyl ring comparing with imines obtained from
aliphatic amines. Some of nitrogen chemical shifts for antipyrine
derivatives are upfield shifted comparing with aliphatic analogs,
which could suggest more advanced proton transfer process. The
nitrogen chemical shift of imine atom in ITFAB dNs = ꢀ79.1 ppm
can verify this suggestion. For aliphatic derivatives without H-bond
Table 1
Results of NMR measurements (1H, 13C, 15N) of investigated compounds in chloroform (if no solvent specified) solution.
4-Amino-antipyrine
SAAP
5-NO2 SAAP
3,5-di-NO2
SAAP
2HNAP
5-Cl SAAP
5-Cl SAAP + 1H
ITFAB
2H5MIT FAB
DMSO
N-10
N-20
C-30
ꢀ259.5
ꢀ199.5
162.0
ꢀ249.1–246.5
ꢀ198.6–199.4
160.3
116.2
149.8
ꢀ247.1–243.5
ꢀ199.9–199.4
159.8
115.0
149.6
ꢀ242.9
ꢀ199.5
ꢀ249.7–246.6
ꢀ199.2–199.1
ꢀ247.9–244.9
ꢀ198.1–199.3
159.9
115.7
149.8
ꢀ232.4
ꢀ198.5
156.13
111.15
146.26
ꢀ249.4
ꢀ198.7
160.6
118.2
152.1
ꢀ245.8
ꢀ199.2
159.7
115.7
151.5
C-40
118.8
138.2
112.1
148.2
116.3
148.9
C-50
NH2
C-100
C-200
C-300
C-400
10-Me
50-Me
10-Me1H
50-Me1H
H-2
ꢀ351.5c1JNH = 78 Hz
b
135.4
122.8
129.0
125.8
37.83
10.21
2.83
2.14
134.3
124.6
129.3
127.3
35.6
10.3
3.17
2.41
134.0
125.1
129.5
127.6
35.3
10.2
3.25
2.45
133.3
125.7
129.7
128.4
34.9
10.3
3.32
2.51
–
134.2
124.8
129.3
127.5
35.5
10.24
3.17
2.39
136.7
127.4
129.85
129.8
34.14
9.68
134.6
124.3
128.9
127.0
35.6
9.9
3.10
2.46
8.33
134.8
125.2
129.6
127.5
35.7
10.3
3.17
2.42
b
–
b
–
b
–
35.5
10.0
3.18
2.45
3.32
2.41
a
H-3
H-4
H-5
H-6
6.95
6.89
7.28
7.35
9.8
7.01
8.17
–
6.87
7.20
6.97
7.38
a
8.88
–
7.88
7.44
7.88
9.81
7.57
a
–
a
8.29
9.87
8.45
9.90
–
7.28
9.73
7.52
9.36
7.57
9.80
2.28
122.8
157.8
122.8
131.0
H-7
10.84
5-Me1H
C-1
b
120.2
160.5
116.7
119.1
119.5
165.9
117.6
127.0
121.6
162.2
137.4
123.7
–
121.1
159.0
118.2
131.5
119.6
158.9
118.56
133.6
138.2
127.3
138.2
129.0
C-2
C-3
C-4
162.3
b
–
b
–
br.
C-5
C-6
b
131.9
132.0
140.2
127.6
137.4
130.9
–
123.6
130.8
124.06
131.69
128.9
129.0
128.2
131.0
b
–
br.
C-7
160.6
158.2
155.4
156.8
158.8
158.5
156.3
154.1
20.5
13.78
ꢀ92.3
5-Me
OHN
Ns
13.33
ꢀ108.5–104.9
14.4
ꢀ105.6–103.3
–
13.0
ꢀ123.4–116.0
13.3
ꢀ104.6–102.0
–
–
–
ꢀ124.1
ꢀ134.2
ꢀ79.1
Nitrogen chemical shifts in CD3CN solution are presented in italics.
a
Unresolved pattern of aromatic protons signals.
Unresolved pattern of aromatic carbons signals.
Results obtained in DMSO solution, since in chloroform solution the NH proton signal was too broad to obtain effective polarization transfer.
b
c

50
W. Schilf et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 109 (2013) 47–54
Table 2
15N and 13C CPMAS NMR data of investigated compounds.
4-Amino-antipyrine
SAAP
5-NO2SAAP
3,5-di-NO2SAAP
2HNAP
5-ClSAAP
2H5MIT FAB
N-10
N-20
C-30
C-40
C-50
ꢀ261.5
ꢀ249.8
ꢀ244.9
ꢀ243.1
ꢀ242.6
ꢀ238.8
ꢀ235.4
ꢀ250.7
ꢀ197.3
ꢀ203.4
159.2a
157.8a
111.9
ꢀ200.3
160.9
ꢀ199.5
156.8
116.2
151.1
ꢀ199.1
160.1
111.4
152.7
ꢀ196.5
155.8 ov.
109.0
ꢀ202.4
157.0
110.9
144.8
ꢀ200.6
159.9
111.9
148.6
120.9 ov.
135.4
119.7
153.7
148.8
147.2
NH2
C-100
C-200
C-300
C-400
10-Me
ꢀ353.2
134.0
122.9 120.9 ov.
130.2
125.3
41.0
35.2
12.0
34.0
9.4
36.1
9.9
33.3
9.0
33.8
9.9
37.7
31.8
8.9
50-Me
Ns
8.7
–
6.8
ꢀ110.5
ꢀ110.1
ꢀ143.8 br.
ꢀ115.2
ꢀ108.5
ꢀ86.3
ꢀ106.7
159.6a
156.4
149.9
C-2
C-7
160.6
156.6
164.1
150.7
164.3
155.8 ov.
161.2
149.8
160.9
153.6
ov – Overlapped signals.
br – Broad signal.
Assignment of some aromatic CH signals is impossible due to overlapping.
a
Assignment can be reversed.
dNs is close to ꢀ60 ppm. The almost 20 ppm upfield shift in ITFAB is
caused by pyrazol-3-one ring. If we consider this effect for the
other antipyrine derivatives it will clear that in all compounds
the proton transfer is less effective than in aliphatic analogs. This
statement is consisting with general rule that in aromatic and
pseudoaromatic imines the proton transfer is weaker than in ali-
phatic analogs. Very interesting is the last compound in Table 1:
2H5MITFAB. In this case the dNs = ꢀ92.3 ppm value suggests the
smallest proton transfer in all investigated compounds. This is
not true, because in this compound two proton-acceptor centers
are present and the observed dNs value is time averaged chemical
shift for two positions: with hydrogen bond and without such
structure. Considering this, and using dNs = ꢀ79.1 ppm value for
non-bonded imine, one can estimate the proper value for hydrogen
bonded structure in this compound as close to dNs = ꢀ105 ppm,
which is in good agreement with the remaining values. For 5-
methyl- substituted derivative (2H5MITFAB) the dNs value close
to those obtain for SAAP was expected.
Basically, the proton position in the H-bonded systems can be
determined by coupling constants analysis too. For the Schiff bases
two couplings: homonuclear 3JHH and heteronuclear 1JNH involving
tautomeric proton can be considered. Unfortunately, this approach
is useful only for the systems where proton is significantly shifted
to nitrogen atom [58]. In considered here compounds the tauto-
meric protons are located on oxygen atoms and mentioned cou-
plings are very small and consequently difficult to measure. In all
investigated compounds the signals of H-7 protons are slightly
broadened singlets without measurable splittings. This broadening
is enough to provide effective proton transfer in GHMBC experi-
ment but not enough to show coupling constants in 2D experi-
ments. In GHSQC experiments we do not observe polarization
transfer from formally OH proton to imine nitrogen atom in all
investigated compounds. Only one successful coupling constant
measurement was done with 4-aminoantipyrine in DMSO solution,
but it do not provide valuable information, it can only confirm sig-
nal assignment. In solid state spectra we do not observe any split-
tings since all the spectra were run with proton decoupling.
The next interesting issue is structural transformation con-
nected with phase change from solution to the solid state. For imi-
nes derived from aliphatic amines with relatively strong H-
bonding, such transformation causes strong proton transfer to
nitrogen atom, which can be monitored by nitrogen chemical shift.
For 5-NO2 derivative nitrogen signal is shifted from dNs = -
ꢀ110 ppm to dNs = ꢀ200.9 ppm and for 3-MeO,5-NO2 compound
from dNs = ꢀ176.1 ppm to dNs = ꢀ207.9 ppm [57]. For investigated
antipyrine derivatives such transformation changes nitrogen
chemical shifts less extensive. Only for 5-NO2 derivative this effect
exceeds about 20 ppm, the most unexpected behavior was found
for naphtyl derivative 2HNAP, where practically no effect of phase
transfer was observed, which means that in both phases the OH
form is dominant one. The Schiff base obtained from naphtylalde-
hyde and methylamine in both chloroform solution and in solid
state exists as NH form (dNs = ꢀ211.6 ppm in solution and dNs = -
ꢀ247.3 ppm in the solid state) [58]. For the 2H5MITFAB sample
in the solid state nitrogen CPMAS spectrum two signals were de-
tected: dNs = ꢀ86.3 ppm and dNs = ꢀ106.7 ppm. Since in this com-
pound two imine sites and only one OH group exist, we can state
that in the solid state the proton transfer between those sites is fro-
zen and consequently signal dNs = ꢀ86.3 ppm should be assign to
non-hydrogen bonded imine and signal dNs = ꢀ106.7 ppm to
hydrogen-bonded site. This is very close to the dNs values in solu-
tion calculated above, it means that the proton position in hydro-
gen bridge is almost the same in both phases. The position of the
proton in hydrogen bridge can be estimate by analysis of carbon
chemical shifts too. Is known the best estimation is provided by
C-2 chemical shift. For antipyrine derivatives the major problem
is with carbon signal assignment in the solid state spectra. Practi-
cally, comparing regular and short contact time spectra we can as-
sign only C-7 signals. The other interesting quaternary carbon
signals (C-30, C-2) are located in very narrow range between 156
and 166 ppm and there is no argument to assign them unmistak-
ably. The position of those signals can lead only to the conclusion
that proton is located close to the oxygen atom (for NH form the
C-2 signals should be downfield shifted to about 170 ppm). The

W. Schilf et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 109 (2013) 47–54
51
remaining quaternary carbon atoms signals (C-40 and C-50) are in
typical position close to 120 and 150 ppm respectively and can
be assigned. Unfortunately, those chemical shifts do not provide
valuable structure information.
dNs = ꢀ102.0 ppm) suggest some structure changes in this posi-
tion, but it cannot be protonation reaction. In the case of full pro-
tonation one can expect much stronger effect. In simple Schiff
bases [53] protonation process shifts nitrogen atom upfield to
dNs = ꢀ180 ppm. Much smaller effect observed for 5-ClSAAP can
be caused be solvent (pKa) effect. The decision which amine nitro-
gen atom was protonated can be done using nitrogen chemical
shifts of those atoms. The chemical shift of N-20 atom is practi-
cally unchanged whereas those for N-10 is shifted downfield
about 12 ppm. In contrast to protonation of imine atom, where
strong upfield effects are observed, the nitrogen chemical shifts
of tertiary amines are much less sensitive on protonation and
additionally this effect can be upfield or downfield [48,54–56].
Since the effect of this range was found on N-10 atom we can
state that this is protonation site.
For the compounds containing more than one nitrogen atom it
is very interesting to decide, where those compounds would be
protonated. For such experiment the 5-ClSAAP derivative was
chosen. The results of NMR measurements of protonated 5-
ClSAAP are presented in Table 1. The analysis of carbon chemical
shifts shows that the biggest differences between neutral and
protonated forms are observed in antipyrine ring and in 100 and
200 position in phenyl ring. It means we can expect that proton-
ation site is in antipyrine ring. To confirm this suggestion we
can compare the chemical shifts of C-7 atom close to possible
protonation site on imine atom. In both neutral and acidic condi-
tions this parameter is completely unchanged. Much better in-
sight into protonation process can be made on the base of
nitrogen chemical shifts. The dNs = ꢀ134.2 ppm value for proton-
ated species (the appropriate value for neutral compounds is
To establish the influence of pH on shape and intensity of UV–
Vis spectrum and pKa values of substituted salicylic aldehydes and
4-aminoantipyrine derivatives a group of four compounds was
chosen (5-ClSAAP, 5-NO2SAAP, 2HNAP and ITFAP).
3
CH3
H2N
OH
4
2
CH3
5'
5
N
4'
N
CH3
1
X
6
7
N
N
CH3
3'
O
1'
N
O
1"
2'
2"
3"
4"
4-{[1-(2-Hydroxy-phenyl)-methylidene]-amino}-
1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one
4-Amino-1,5-dimethyl-2-phenyl-1,2
-dihydro-pyrazol-3-one
X=H
X=5-NO2
SAAP
5-NO2SAAP
4-Aminoantipyrine
X=3,5-diNO2 3.5-diNO2SAAP
X=5-Cl
5-ClSAAP
Antipyrine
OH
Antipyrine
Antipyrine
N
N
N
OH
CH3
2HNAP
2H5MITFAP
Antipyrine
N
Antipyrine
N
ITFAP
Fig. 1. Compounds under investigations.

52
W. Schilf et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 109 (2013) 47–54
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
long-wave band from 364 nm in pH = 8 to 383 nm in pH = 16 and
(b) ITFAP
pH=8
to 425 nm in pH = 23 was observed (Fig. 2a). Generally all investi-
gated compounds show similar changes of UV–Vis spectra. In alka-
line solution we observe a batochromic shift, whereas in acidic
solution there is a hypsochromic shift present. In case of ITFAP
derivative there are no changes of the spectrum in alkaline solution
(Fig. 2b).
(a) HNAP
pH=8
pH=16
pH=23
pH=16
pH=23
(a)
0.8
0.6
0.4
300
400
[nm]
500
300
400
[nm]
500
λ
λ
measured data
calculated data
0.2
0.0
Fig. 2. Absorption spectra of HNAP (a) and ITFAP (b) in different pH.
R2 = 0.908
pKa = 7.59 0.03
14
Investigated compounds, 5-ClSAAP, 5-NO2SAAP, 2HNAP and IT-
FAP in acetonitrile:methanol (9:1) solution exhibit strong changes
in position and intensity of UV–Vis spectra, when the pH is chan-
ged from basic to acidic. For example for 2HNAP a shift of the
6
8
10
12
16
pH
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(b)
0.7
(a)
0.6
0.5
0.4
0.3
0.2
0.1
0.0
measured data
calculated data
R2 = 0.99
pKa = 18.63 0.02
15
16
17
18
19
20
21
22
23
24
300
350
400
450
pH
λ
[nm]
(c)
0.8
0.6
0.4
0.2
0.0
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
(b)
measured data
calculated data
R2 = 0.947
pKa1 = 8.59 0.20
pKa2=16.05 0.12
6
7
8
9
10
11
pH
12
13
14
15
16
17
300
350
400
450
500
λ
[nm]
Fig. 4. Spectrophotometric titration curves – A vs pH curves for: (a) 5-NO2SAAP at
k = 385 nm in pH range 8–16, (b) 5-NO2SAAP at k = 433 nm in pH range 16–23, and
(c) ITFAP at k = 343 nm in pH range 8–16.
Fig. 3. Titration spectra of 5-ClSAAP in mixture acetonitrile:methanol (9:1) in pH
range: (a) 8–16 and (b) 16–23.

W. Schilf et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 109 (2013) 47–54
53
Table 3
diNO2 derivative, and for naphtyl derivative we observe opposite
effect, in solid state proton is shifted less to nitrogen atom then
in chloroform solution.
pKa values with uncertainties obtained for 5-ClSAAP, 5-NO2SAAP, 2HNAP and ITFAP
in mixture acetonitrile:methanol (9:1).
Compound
pKa1
pKa2
Concluding, we can state that the antipyrine substituent is the
most important part of the molecule under study. The electron ef-
fects created by this substituent are so strong that can minimize
influence of acidity of OH group on the H-bond structure. Addition-
ally, this substituent successfully prohibits all structural changes
which could promote the proton transfer from oxygen to nitrogen
site in the solid state, which leads to the situation that in both
phases we observe almost the same structure.
On some quantitative level the protonation site can be define on
the base of pKa value measurements. In all investigated compounds
the pKa values in acidic solution are very close one to another. This
suggest that that protonation takes place on the fragment of the
same structure. In the case of protonation on imine atom the intra-
molecular hydrogen bond of different structure should modify this
parameter. The amine sites in antipyrine ring are almost equal in
all derivatives and should have similar basicity and consequently
similar pKa values. Unfortunately, on the base on available data
in is impossible to decide which amine atom in protonated.
5-ClSAAP
5-NO2SAAP
2HNAP
7.76 0.02
7.59 0.03
8.05 0.04
8.59 0.20
20.03 0.06
18.63 0.02
20.35 0.06
16.05 0.12
ITFAP
UV–Vis spectra of these molecules were recorded in pH range
8–23 (Fig. 3a and b). Observed spectral changes suggest the pres-
ence of two acid–base equilibria for all investigated compounds.
The complex character of the acid–base equilibria are consistent
with the changes of absorption as a function of pH, where for se-
lected wavelength there are inflections visible, suggesting presence
of minimum three protolytic forms. For 5-ClSAAP, 5-NO2SAAP and
2HNAP one equilibria was determined in acidic solution (Fig. 4a)
and the second one in alkaline solution (Fig. 4b), whereas for ITFAP
both equilibria were established in acidic solution (Fig. 4c). This is
the result of the compound structure. ITFAP is not a salicylalde-
hyde derivative. The acid–base equilibria present in alkaline solu-
tion results from dissociation of the phenolic substituent in the
salicylaldehyde group, whereas the one present in acidic solution
is associated with the aminoantipyrine group. Analyze of the pH-
spectroscopic titration curves indicates, that in higher concentra-
tion of acid there is not only protonation of the compound present,
but presumably also hydrolysis of the C–N bond. This hinders the
quantitative analysis of these changes.
Values of the first dissociation constant of the investigated mol-
ecules, 5-ClSAAP, 5-NO2SAAP, 2HNAP and ITFAP differ insignifi-
cantly in range 7.59–8.59 (Table 3). For 5-ClSAAP, 5-NO2SAAP
and 2HNAP pKa1 value reflects the alkaline properties of the mole-
cule, while pKa2 value reflects the acidic properties of the molecule.
ITFAP derivative containing two aminoantipyrine groups, both
capable of proton acceptance, exhibit stronger alkaline properties
than their derivatives with salicylic aldehyde group. Comparison
of the pKa2 values of salicylic aldehyde derivatives suggests that
the presence of nitro group (5-NO2SAAP) in para position to hydro-
xyl group increases the acidity of the phenolic group (pKa2 = 18.63)
in relation to compounds containing chloride atom (pKa2 = 20.03)
in this position or being 1-hydroxynaftoic aldehyde derivative
(pKa2 = 20.35) (5-ClSAAP and 2HNAP).
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