In situ observation of ninhydrin and phenylhydrazine reaction in solution by FTIR

Article abstract of DOI:10.1016/j.saa.2013.05.002

(Graph Presented) The current work was performed for hydrazone formation reaction of ninhydrin with phenylhydrazine in chloroform by using "background defining method" in FTIR liquid cell. The method allowed following the consumption and formation of the

Full text of DOI:10.1016/j.saa.2013.05.002

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 297–301
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
In situ observation of ninhydrin and phenylhydrazine reaction in
solution by FTIR
⇑
Onur Turhan , Elif Tezbaßsaran
Balikesir University, Faculty of Arts and Sciences, Department of Chemistry, Cagis, 10145 Balikesir, Turkey
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
ꢀ
ꢀ
ꢀ
ꢀ
Hydrazone formation reaction of
ninhydrin and phenylhydrazine was
investigated.
In situ observation of the reaction is
realized by using background
defining method.
The reaction performed to two
different mol ratios (1:1, 1:3) of
reagents.
Both run are resulted in same product
(1,2,3-tris-phenylhydrazonoindan).
a r t i c l e i n f o
a b s t r a c t
Article history:
The current work was performed for hydrazone formation reaction of ninhydrin with phenylhydrazine in
chloroform by using ‘‘background defining method’’ in FTIR liquid cell. The method allowed following the
consumption and formation of the reagent and product at the same time. Negative absorption bands are
based on reagent consumption and positive absorbances are based on product formation. The method
was applied for two different mol ratios (1:1, 1:3) of reagent. Both ratios resulted in same product
(1,2,3-tris-phenylhydrazonoindan). The results showed that, phenylhydrazine react with all of the car-
bonyl groups in ninhydrin at both run.
Received 30 January 2013
Received in revised form 30 April 2013
Accepted 2 May 2013
Available online 13 May 2013
Keywords:
Ninhydrin
Background defining
Hydrazone formation
In situ time dependent FTIR
Ó 2013 Elsevier B.V. All rights reserved.
Introduction
been studied [6]. It is reported that ninhydrin gives Ruhemann
purple not only with amino acids with some aliphatic amines
Ninhydrin is a well known common coloration reagent for
amino acids. Since ninhydrin’s first synthesis in 1910 by Ruhe-
mann, [1] many ninhydrin derivatives were synthesized and chem-
ical properties were investigated [2]. As shown in Scheme 1
ninhydrin can be considered as a tricarbonyl compound due to
its equilibrium with indane-1,2,3-trione [3].
The well known reaction of ninhydrin is generating Ruhemann
purple with amino acids [4,5]. After Ruhemann’s synthesis, the
reaction of ninhydrin with some amine and amide derivatives have
too. On the Ruhemann reaction ninhydrin reacts with amines
and amino acids with its hydrated carbonyl at 2 position. The
source of N atom in Ruhemann purple structure is the amino acid
or primary amines. The general mechanism for the ninhydrin reac-
tion is illustrated in Scheme 2. Ninhydrin (I) is in equilibrium with
1,2,3-indantrione which forms a imine (II–III) with the amino acid
and primary amines. The formed ketimine (V) yielding the alde-
hyde and an intermediate amine (2-amino-l,3-indandione, VI),
which was isolated by Ruhemann. Condensation of this intermedi-
ate amine with another molecule of ninhydrin follows to Ruhe-
mann’s Purple (VII) [5]. Ninhydrin and derivatives are also
important compounds for criminal workouts because of their fin-
gerprints visualization properties [7,8].
⇑
Corresponding author. Tel.: +90 2666121200; fax: +90 2666121215.
E-mail addresses: oturhan@balikesir.edu.tr (O. Turhan), eliftezbasaran@gmail.
com (E. Tezbaßsaran).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.05.002

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O. Turhan, E. Tezbaßsaran / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 297–301
used [15]. The method, that we developed previously, let us to
O
O
understand whether a reaction carried out or not and how the
reaction is carried out in the reaction media. The method also pro-
vides information about functional group transformation.
On the FTIR studies scanning background is necessary to elimi-
nate the vibrations due to the background material such as KBr for
solids and solvent for solution. In our previous study we have
found that background scan can also be performed for a solution
of a reagent at the beginning of a reaction as well [15]. In this study
background is recorded at the preliminary of the reaction. Elimina-
tion of the absorption bands due to the reagents and the solvent
will produce a clearer spectrum which shows only the absorption
bands due to the changes of the reaction media. It is also possible
defining the background at any time of the reaction and this allow
us to eliminate all the peaks arising from any component or even
product formed until that time and let us follows any changes after
the background recording time.
OH
OH
O
+ H2O
O
O
ninhydrin
(2,2-dihydroxy-1,3-indandione)
indan-1,2,3-trione
Scheme 1. Equilibrium of ninhydrin with indane-1,2,3-trione.
The reaction of ninhydrin with some aromatic amines produced
2-hydroxy-2-arylamino-1,3-indandione derivatives [9]. Further-
more ninhydrin formed cyclization products with some of electron
releasing groups (hydroxy, methoxy and amino) substituted aro-
matic amines [10] (Scheme 3).
Although the reaction of ninhydrin with amino acids and
amines were investigated by many researchers, the reactions of
ninhydrin with hydrazine derivatives are scare [11–14]. Hydra-
zines have similar features with primary amines by some diversity.
Both functional groups have nucleophilic behaviour but amines are
much more alkali in characteristics while hydrazine is much more
nucleophilic. While ANH2 group in hydrazine is bonded with an-
other electronegative N atom, it is directly bonded to the C atom
in amines.
The method was successfully applied in our previous studies
about the identification on imine formation reaction of some aro-
matic aldehyde with aniline [15]. The method was also used for a
kinetic study of benzaldehyde and aniline reaction in chloroform
[16].
Experimental
The aim of this study is in situ investigation of the reaction of
ninhydrin with phenylhydrazine. For this purpose background
defining FTIR method which is described in our previous study, is
FTIR spectra were collected using Perkin–Elmer Spectrum 65
instrument using 0.015 mm path length CaF2 liquid cell.
R
COOH
O
O
H
O
NH
2
O
O
N
O
O
O
a
H
OH
OH
- CO
2
II
N
C
R
H N CH
R
2
2
H
O
H O
2
H
H O
I
2
b
IV
N
R
H
O
III
O
O
O
O
O
+ I
+
H
H O/ H
H
N
2
N
NH
+
2
CHR
H
- RCHO
O
O
O
V
VI
VII
Ruhemann purple
Scheme 2. A model reaction of Ruhemann purple from ninhydrin and amino acids.
O
O
O
O
R
R
2
3
OH
NH
OH
NH
N
R
1
N
HO
R =R =R =H
1
2
3
R =Cl, R =R =H
X
1
2
3
X= OH, OCH
3
R =OH, R =R =H
2
1
3
Scheme 3. The products of ninhydrin with some aromatic amines.

O. Turhan, E. Tezbaßsaran / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 297–301
299
1:1 and 1:3 for the first and second run. 1:1 mol ratio is described
as first run and 1:3 mol ratio is second run in the text for both
in situ and laboratory conditions. Observed FTIR spectra of both
run have shown in Figs. 1 and 2 respectively. The characteristic
symmetric and asymmetric stretching bands of ninhydrin carbon-
yls appeared at 1755, 1725 cmÀ1 and the characteristic phen-
ylhydrazine aromatic vibration at 1601 cmÀ1 and ANH2
vibrations at 3382, 3355 cmÀ1 in chloroform.
3
2
As expected, the decreasing vibrations in the spectra (below the
absorbance line) exactly match in frequency with the reagents
vibrations in the solutions, indicating a concentration decrease
due to consumption of the reagents. On the other hand, the
increasing vibrations (above the absorbance line), which are at
the same frequency with the product, indicate formation of a
new compound. Newly formed products C@N vibration is appeared
at 1532 cmÀ1 (Figs. 1a and 2a) [17].
Scheme 4. Hydrazone formation reaction of ninhydrin with phenylhydrazine.
Definition of the method
1. Appropriate concentration of ninhydrin (0.05 M) and phen-
ylhydrazine (0.05 or 0.15 M) solutions were prepared in
chloroform and FTIR spectra of each solution were recorded
for comparison.
2. The equal volume of reagent solutions were mixed and
immediately scanned in same CaF2 liquid cell.
3. The initially scanned spectrum of the mixture was recorded
as a background for the latter real time scans.
4. Latter spectra were collected in specific time intervals for
both run.
Ordinary synthesis of 1,2,3-tris-phenylhydrazonoindan
To compare the real time monitored FTIR spectra with the prod-
uct absorption bands, reaction also be carried out under normal
laboratory condition. The reaction is performed for two different
mol ratios as 1:1 and 1:3. For first run 0.01 mol ninhydrin and
0.01 mol phenylhydrazine were mixed in 20 mL chloroform at
room temperature. After 24 h precipitate was filtered off and
recrystallized from benzene. The same reaction is also carried out
with 0.03 mol phenylhydrazine and 0.01 mol ninhydrin. (Scheme
4) The obtained products in both run are analyzed by 1H NMR,
13C NMR and MS. The comparison of spectral data clearly proved
that in both run the same product (1,2,3-tris-phenylhydrazonoin-
dan) have been obtained (mp: 211 °C). 1H NMR (dDMSO, d, ppm)
7.64–7.67 (d, 2H), 7.62–7.64 (d, 2H), 7.50–7.53 (t, 6H), 7.40–7.44
(d, 6H), 7.06–7.12 (t, 3H), 12.74–12.79 (s, broad, @NANHA). 13C
NMR (CDCl3, d, ppm) 183.7 (C@NANH), 142.7, 134.1, 129.7,
128.5, 123.7, 119.1, 114.6. MS m/z: 433 (M+2), 263, 157, 109, 93, 78.
5. Increasing and decreasing peaks were compared with the
reagents and product.
Chloroform is used as solvent because it has no limitation over
1250 cmÀ1 in middle infrared region and allowed us to follow the
conversion of the functional groups.
Hydrazone formation reaction of ninhydrin and phenylhydr-
azine in chloroform (Scheme 4) was investigated by using back-
ground defining of the beginning of the reaction. Due to the
aforementioned background defining method, all absorption bands
seen in Fig. 1c and d can be ignored by FTIR software. This elimina-
tion provides to monitor, any change in reaction media by getting
FTIR spectrum in particular time interval. In situ obtained spectra
of reaction media are shown in Figs. 1b and 2b.
Result and discussion
In this study, an easy application of FTIR spectroscopy for the
observation of hydrazone formation in
demonstrated.
a liquid cell was
To examine the reaction of ninhydrin and phenylhydrazine the
described method was applied in two different mol ratios of the
reagents. Mol ratios of the ninhydrin and phenylhydrazine were
We have observed that all the carbonyl groups in ninhydrin re-
act with phenylhydrazine to produce hydrazone. In the both run no
1532
1532
1255
1599
1489
(a)
1255
1489
1601
1718
(b)
A
1496
1601
3382
3355
1725
(c)
(d)
1755
1598
4000
3500
3000
2500
2000
1500
1250
Wave numbers (cm-1)
Fig. 1. FTIR spectra of; (a) isolated product, (b) in situ reaction media (1:1), (c) phenylhydrazine, (d) ninhydrin in chloroform (a, c and d spectra were arbitrarily displaced in
vertical for clarity).

300
O. Turhan, E. Tezbaßsaran / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 297–301
1532
1532
1490
1599
(a)
(b)
A
1601
1496
3355
3382
(c)
(d)
1725
1755
4000
3500
3000
2500
2000
1500
1250
Wave numbers (cm-1)
Fig. 2. FTIR spectra of; (a) isolated product, (b) in situ reaction media (1:3), (c) phenylhydrazine, (d) ninhydrin in chloroform (a, c and d spectra were arbitrarily displaced in
vertical for clarity).
Table 1
The peak heights of C@O and C@N (Abs) of both run.
Time (min)
Absorbance values
First run
Second run
1725 cmÀ1 (C@O vibs.)
1532 cmÀ1 (C@N vibs.)
1725 cmÀ1 (C@O vibs.)
1532 cmÀ1 (C@N vibs.)
3
6
9
À0.0123
À0.0182
À0.0243
À0.0311
À0.0397
À0.0449
À0.0493
À0.0544
À0.0599
À0.0649
À0.0693
À0.0731
À0.0763
À0.0797
À0.0850
À0.0907
À0.0935
À0.0958
À0.098
0.0044
0.0082
0.0123
0.0180
0.0225
0.0335
0.0471
0.0581
0.0706
0.0822
0.0934
0.1034
0.1131
0.1219
0.1294
0.1327
0.1390
0.1447
0.1504
0.1549
0.1610
0.1691
0.1756
0.1812
0.187
À0.0023
À0.0142
À0.0265
À0.0441
À0.0535
À0.0624
À0.0716
À0.0809
À0.0881
À0.0972
À0.1045
À0.1121
À0.1196
À0.1249
À0.1310
À0.1360
À0.1404
À0.1437
À0.1471
À0.1502
À0.1545
À0.1591
À0.1621
À0.1649
À0.1672
À0.1691
À0.1722
À0.1735
À0.1763
0.0043
0.0206
0.0459
0.0687
0.0934
0.1182
0.1447
0.1720
0.1904
0.2150
0.2349
0.2555
0.2770
0.2923
0.3104
0.3257
0.3403
0.3498
0.3612
0.3712
0.3851
0.3983
0.4081
0.4169
0.4245
0.4307
0.4411
0.4478
0.4555
12
15
18
21
24
27
30
33
36
39
42
45
48
51
54
57
60
65
70
75
80
85
90
100
110
120
À0.1000
À0.1021
À0.1054
À0.1077
À0.1101
À0.1123
À0.1138
À0.1168
À0.1192
À0.1207
0.1914
0.2016
0.2116
0.2181
carbonyl stretching were observed in product FTIR spectra. The re-
sult showed that there was no selectivity for phenylhydrazine in
any carbonyl of ninhydrin. Ninhydrin usually reacts with amines
and amino acids with its hydrated carbonyl [6–8]. We have exam-
ined this phenomenon for ninhydrin and different mol ratios of
phenylhydrazine by expecting different product. We have found
that even ninhydrin starts to react from its centre carbonyl with
phenylhydrazine the reaction also continuous at the 1 and 3 posi-
tions. The tris-hydrazone was the expected product for the second
run due to the mol ratios. However the first run produced the same
product even for 1:1 mol ratios.
In situ monitoring of the reaction media is more useful in this
kind of situation while only the change of the reagents and
products have been observed. When in situ FTIR spectra in Figs.
1b and 2b examined carefully, it is clear that both reactions are
proceed in same way. It may be suggested that hydrazone forma-
tion may start from hydrated carbonyl of ninhydrin; however oc-
curred 2-hydrazono-1,3-indandione could be more reactive than
ninhydrin itself. So both run may be produced the tri-hydrazone
product.
In the in situ FTIR spectra (Figs. 1b and 2b) the absorption band
intensities are directly related with the consumption of the

O. Turhan, E. Tezbaßsaran / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 297–301
301
0.25
0.2
triplicate. Both run were produced the same product with different
reaction rates. As can be seen from Figs. 3 and 4 the increase in
product concentration in second run is higher than first run by
the time.
0.15
0.1
1532 cm-1
1725 cm-1
Conclusions
0.05
0
Defining background at the beginning of the reaction provides
an easy observation of the changes during the reaction in the FTIR
spectra. The spectra of product can be obtained without isolating it
from the reaction mixture due to the positive absorption whereas
the absorption bands of reactants are negative. One of the main
advantages of used method is observation of reagent vibrations
and product vibrations in different side of the absorbance line.
The other one is unreacted reagent vibrations in media are not
causing any problem due to background defining at the beginning
of the reaction.
Time (min)
0
20
40
60
80
100
120
-0.05
-0.1
-0.15
Fig. 3. Time to peak height (Abs) plots of the first run (1:1) reaction.
The results showed that all carbonyl groups in ninhydrin
reacted with phenylhydrazine to produce hydrazone. The tris-
hydrazone product was observed in the both run. There was no
selectivity of any carbonyl of ninhydrin for phenylhydrazine while
amines select hydrated carbonyl.
0.5
0.4
0.3
0.2
Acknowledgment
1532 cm-1
0.1
This study was financially supported by Balikesir University
Scientific Research Project Units (Project 2010/25).
1725 cm-1
time (min)
0
0
20
40
60
80
100
120
References
-0.1
-0.2
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heights of the C@O vibration at 1725 cmÀ1and the increase in
C@N vibration at 1532 cmÀ1 for both run in the same time intervals
are summarized in Table 1. Negative intensities are originated from
ninhydrin consumption and also positive intensities are originated
from the production of tris-hydrazone product.
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