Photosonoelectrochemical analysis of Lawsonia inermis (henna) and artificial dye used in tattoo and dye industry

Article abstract of DOI:10.1016/j.jphotochem.2018.04.026

Photosonoelectrochemical (PSEC) analysis of Lawsonia inermis, lawsone and ?-Phenylenediamine were investigated in ethanol to understand the degradation mechanism and harmful byproducts. To simulate the operating conditions of the tattoo ink, dye solutions

Full text of DOI:10.1016/j.jphotochem.2018.04.026

Accepted Manuscript
Title: Photosonoelectrochemical analysis of Lawsonia inermis
(henna) and artificial dye used in tattoo and dye industry
Authors: T. Chandrakalavathi, V. Sudha, M. Sindhuja, S.
Harinipriya, R. Jeyalakshmi
PII:
S1010-6030(18)30221-1
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.04.026
JPC 11241
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
16-2-2018
8-4-2018
10-4-2018
Please cite this article as: T.Chandrakalavathi, V.Sudha, M.Sindhuja, S.Harinipriya,
R.Jeyalakshmi, Photosonoelectrochemical analysis of Lawsonia inermis (henna) and
artificial dye used in tattoo and dye industry, Journal of Photochemistry and
Photobiology A: Chemistry https://doi.org/10.1016/j.jphotochem.2018.04.026
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1
Photosonoelectrochemical analysis of Lawsonia inermis (henna) and artificial dye used in tattoo and dye
industry
T. Chandrakalavathi1, V.Sudha1, M.Sindhuja2,3, S.Harinipriya1,2*, R. Jeyalakshmi1
1Department of Chemistry, SRM Institute of Science and Technology, Kattankulathur 603203, India
2*Corresponding Author, Electrochemical systems Laboratory, SRM Research Institute, SRM Institute
of Science and Technology, Kattankulathur
3Department of Physics and Nanotechnology, SRM Institute of Science and Technology,
Kattankulathur, 603203, India.
Graphical Abstract
Lawsone

2
PPD
Highlights


Lawsonia inermis donot undergo degradation upon exposure to PSEC perturbation
commercial lawsone undergo oxidation upon PSEC perturbation forming ethoxy substituted
napthaquinone


PPD, an artificial dye resulted in the formation of amino substituted cyclobutadiene upon PSEC
perturbations.
Formation of carcinogenic byproducts by lawsone and PPD respectively demonstrated the harmfulness
in utilizing these artificial dyes as colour enhancing agent to henna by dye and tattoo industries.
Abstract.
Photosonoelectrochemical (PSEC) analysis of Lawsonia inermis, lawsone and ƥ-Phenylenediamine
were investigated in ethanol to understand the degradation mechanism and harmful byproducts. To
simulate the operating conditions of the tattoo ink, dye solutions were exposed to appropriate
sonication, UV irradiation and electrochemical perturbations. Analyses of the dyes were done before
and after PSEC perturbations employing UV-Vis, FTIR, HPLC, 1H NMR and 13C NMR studies and
degradation pathway is provided. The PSEC studies inconjunction with the spectroscopic and HPLC
analysis identified formation of byproducts such as 3-ethoxy naphthaquinone from lawsone and 1-
Amino cyclobutadiene from ƥ-Phenylenediamine demonstrating the harmfulness of utilizing these
dyes over henna by tattoo and hair dye industries.

3
Keywords: Lawsonia inermis; lawsone; ƥ-Phenylenediamine; Cyclic Voltametry and UV-Visible;
FTIR; 1H NMR spectroscopy; sonication.
E-mail: harinipriya.s@res.srmuniv.ac.in
1. Introduction:
Natural dyes are extensively used in cosmetic industries[1] in the form of biodegradable commodities
due to its non-toxic, non-carcinogenic and biodegradable nature. For centuries, henna had been used
by cosmetic and tanning industries to dye skin, hair, fingernails, silk, wool, and leather [2-4]. The
coloring property of henna (Lawsonia inermis) was due to lawsone[5]. Several studies were
reported[3] on the antimicrobial activity and effect on hair growth and invivo injury recovery[6] by
henna. The utility of commercial lawsone (2-hydroxy-1,4-naphthoquinone) ranges from traditional
tattoo ink (henna) to medicinal remedies. It was used as artificial-tanning, hair colorants[7], antifungal
agent[8,9] and antioxidant in rubber industry[10]. In general, henna is harmless except for people with
glucose-6-phosphate dehydrogenase deficiency[11]. Most of the commercial henna body art products
were mixtures of henna and other dye compounds such as silver nitrate, carmine, pyrogallol, disperse
orange dye, ƥ-phenylenediamine (PPD) and chromium[12,13]. PPD is a skin sensitizer which causes
Contact Allergy Dermatitis [14,15]. Pure henna was a weak sensitizer, but the addition of PPD made
tattoos darker and long-lasting. Thus obtaining black henna, by mixing henna with various
concentrations of PPD resulted in contact sensitization to PPD[16,17]. Ultrasound was found to be an
attractive advanced oxidation technology for the degradation of hazardous organic compounds[18]. In
general the combination of photocatalysis and ultrasound was considered to enhance the degradation
rate [19-20]. Sonoelectrochemical technique is not new to scientists as most of the electroanalytical
and electrosynthesis utilize sonoelectrochemistry for homogeneous electrochemical reaction and
uniform mass transport to the electric double layer from bulk of the electrolyte solution[21]. Although
several techniques and methodology had been followed by researchers to study and understand the
degradation mechanism of artificial dyes, none so far on PSEC perturbations had been investigated.
As the tattooing machine utilizes electric or electromagnetic impulses to perform tattoo art on human

4
skin and the machine vibrates during the process, simulating these conditions via PSEC perturbations
can provide a realistic approach to identify the degradation products. Most of the tattoo inks utilize
solvents such as ethanol, water, methanol or mixture of water and ethanol/water and methanol, hence
we conducted the PSEC experiments in 50% ethanol solution. 3% of solar radiation constitutes UV
rays (42 W/m2), the maximum threshold for human skin tolerance towards UV radiation received by
earth is 25%. Thus in the current study, the dyes were exposed to 10.4mW/cm2 of UV radiation. Thus
the major objective of the present work are PSEC analysis of henna, lawsone and PPD in ethanol to
understand the mechanistic pathway of their degradation, spectroscopic analysis of the dyes before
and after PSEC treatment to understand the degradation products formed and the mechanism of
degradation, HPLC and 13CNMR analysis of the products formed and to insist on the usage of henna
rather than other artificial dyes in tattoo and dye industries.
2. Experimental details:
All reagents were of analytical grade and used without further purification. Herbal henna was
purchased from Aravindh Herbal Labs (P) Ltd., Rajapalayam, India. 2-hydroxy -1,4-napthoquinone
(Lawsone) was purchased from Sigma Aldrich India Pvt. Ltd. PPD and ethanol were purchased from
loba chemie pvt. ltd., India.
Photolytic activity
Photolytic activity of 0.1% and 0.5% concentrations of Lawsonia inermis, lawsone and PPD were
tested and compared for their degradation. These solutions were exposed to UV light (125W Hg arc,
10.4 mW/cm2) under constant sonication (40 kHz) for 3hrs.
Electrochemical studies
Zahnner-pp 211 electrochemica1 workstation was used for electrochemical analysis. Cyclic
Voltammetry(CV) and Linear Sweep Voltammetry(LSV) were used to study the mechanism of
electron transfer in Lawsonia inermis, lawsone and PPD. The electrochemical experiments were
carried out using three electrode assembly with Ag/AgCl electrode as reference electrode, Pt wire as
counter electrode and glassy carbon as working electrode. The concentration range of henna, lawsone

5
and PPD were 0.1% and 0.5% as these were the concentrations used in artificial dye. The potential
range was -1 to +1V, at scan rates of 25, 50 and 100mV/s.
Spectroscopic studies
1
13
UV-Vis, FTIR, H NMR and C NMR analysis were studied to investigate the byproducts formed
after PSEC perturbations. The UV–Vis absorption spectra of the samples were measured using
SPECORD-210 UV spectrophotometer at room temperature in the wavelength range of 190–1100 nm
in ethanol. The samples were analyzed by BRUKER ALPHA E&T FTIR spectrophotometer using
KBr pellette. The absorption of electromagnetic radiation is in the wave number range of 400-4000
1
cm-1. Solution state H NMR spectra was recorded by Bruker 500 MHz standard bore (SB) NMR
13
spectrometer equipped with BBO probe head, while solid state C NMR Cross Polarization Magic
Angle Spinning (CPMAS) spectra were recorded on a Bruker 500 MHz wide bore (WB) NMR
spectrometer equipped with a solid state MAS probe head before and after electrochemical
experiments to understand the structural shift due to perturbations.
HPLC experiments:
HPLC-DAD Column Xbridge-C18 with mobile phase of water-ethanol (50-50) mixture, at a flow rate
of 1 ml/min was used to perform HPLC studies. The injection volume is 10ul. The column oven
temperature was maintained at 35oC. Different wavelengths were specified for lawsone and PPD.
Lawsone at 280 nm, PPD at 242 nm. These wavelengths allow a sufficient sensitivity of detection for
the determined compounds to be obtained.
3. Results and discussion
3.1 Electrochemical studies
The electrochemical studies of the sample were done using three electrode assembly (i) during UV
irradiation and sonication and (ii) after UV irradiation and sonication.
3.1.1 Cyclic Voltammetry
a) Lawsonia inermis
The absence of redox behavior for 0.1% and 0.5% Lawsonia inermis in ethanol (Figs.1a&b), indicated
that the dye component (lawsone) was not freely available to be extracted in ethanol due to the
presence of other constituents such as chlorophyll. Thus degradation of lawsone, the basic colorant in

6
Lawsonia inermis was not possible via UV-irradiation, sonication and electrochemical treatment,
hence the dye remain longer on skin without degradation and harmful effects.
b) Lawsone
Sonicating the ethanolic dye solution reduced the particle size with increase in the reactivity of
lawsone with ethanol. Therefore oxidation peak at 0.5 V was observed (Figs 1c&d) and the peak
current increased with scan rate. The oxidation peak could be attributed to the formation of quinonoid
ring in the solution due to electrochemical perturbation. As peak current is directly proportional to
concentration of the reactive species and square root of the scan rate, with scan rate the peak current
increases. Due to oxidation of lawsone, concentration of quinonoid species in solution increases
leading to increased peak current. Thus due to PSEC perturbation lawsone is oxidized to quinonoid
compound as supported by spectroscopic and HPLC analysis in the following sections.
The diffusion coefficient of lawsone could be calculated using Randles-Sevick equation given below.
ip= Kn3/2 A D1/2 C ν1/2
(1)
where ip represent peak current in amps, K being 2.69x105 C mol-1 V-1/2, n denoted number of electrons
transferred in the redox reaction (n=1), A depicted area of electrode in cm2, D indicated the diffusion
coefficient in cm2/s, C being the concentration of lawsone (mol/cm3) and ν represent the scan rate in
mV/s. On substituting ip values at different scan rates (25,50 and 100mV/s), for 0.1% and 0.5%
lawsone, D values were calculated using eqn (1). At low concentration the diffusion coefficient was
higher as can be seen from Fig 2a. In addition, the peak current ip, for the oxidation peak is lower at
all scan rates for 0.5% lawsone after PSEC experiments (cf. Fig 2b). Thus at lower concentrations, the
degradation of lawsone was faster in comparison to the higher concentrations. The reason for this
facile degradation at lower concentration is the surplus availability of solvent which acts as
chromophore and initiates degradation of lawsone.
c) PPD
In general, PPD easily losses proton from amine group in water and form conjugated product. Since
ethanol is less polar than water, PPD gets involved in H-bond instead of conjugated product formation.

7
This could be attributed to aromatic ring opening during sonication and UV irradiation. The redox
behavior was not seen due to the formation of electro inactive byproducts via aromatic ring opening
1
(cf. Figs. 1e&f) and rearrangement reaction as can be deduced and supported by FTIR, H NMR,
HPLC and 13C NMR analysis (cf.section 3.2). Although redox reaction is not seen, electrochemical
perturbations facilitates the reaction between the byproducts formed due to UV irradiation and
sonication leading to further degradation of the intermediates into substituted dienes and conjugated
aromatic diamines as discussed in following sections.
3.1.2. Linear Sweep Voltammetry (LSV)
Lawsone depicts a significant peak current with the concentration of the dye in LSV(Fig 3). In PPD
the intensity of the peak increased with concentration(Fig 3). The diffusion coefficient of lawsone for
all scan rates at 0.1% and 0.5% concentrations were calculated as mentioned earlier and plotted (cf.
Figure 4 a and b). Analogous to CV analysis lawsone demonstrated peak current proportionality to
scan rate (cf. section 3.1 and Fig 4b).
a) Lawsone
Figs 3 (a & b) show an anodic oxidation peak for 0.1% and 0.5% of lawsone in ethanol. The effect of
scan rate on the PSEC process was evaluated (25,50 and 100mV/s). The purpose of the experiment
was to determine whether the oxidation of species in solution was limited by electron transfer or
anolyte diffusion[22] process. The results indicate that 0.1% lawsone had higher peak current than
0.5% of lawsone. Each curve has the same form but it was apparent that the total current increased
with increasing scan rate. Due to sonication the peak current at 0.1% is as significant as 0.5%.
Sonication helps in homogeneous supply of electroactive species from bulk of the solution to the
electrical double layer (where the electron transfer occurs). Even though the amount of solute
(lawsone) is less in 0.1%, sonication of the solution during electrochemical analysis provides
continuous supply of solute to the interface. On the contrary, at 0.5% as the solute is saturated in the

8
solution, sonication creates disturbance to the mass transfer from bulk to interface and leads to
undesired electrochemical activity such as hydrogen evolution and oxygen reduction reactions.
½
Figs 4 (a & b) depict D1/2 vs ν and i vs ν results of 0.1% and 0.5% of lawsone . Here the peak current
was high at 0.1% concentration than 0.5% due to the reduction of lawsone particle size during
sonication. Thus lawsone was highly active at low concentration with high ion transfer rate compared
with higher concentration. Thus CV and LSV data of lawsone demonstrate that, at 0.1% concentration
lawsone is electro active and the peak current is higher than 0.5% concentration. Hence no products
and ring open reaction occurred at 0.5% due to solubility effect as discussed above and supported by
spectroscopic studies (cf.section 3.2).
b) PPD
Figs 3 (c & d) represent the LSV of 0.1% and 0.5% PPD in ethanol. The purpose of the experiment
was to determine the oxidation of PPD upon PSEC perturbations. PPD has no oxidation peak but only
semiconductor behavior in ethanol due to quenching effect by the solvent (cf.section 3.2). As discussed
in CV studies, PPD undergo further degradation of the conjugated aromatic diamines into dienes. This
reaction does not show oxidation/reduction profile, instead utilizes the electrochemical impulses to
initiate degradation process via aromatic ring opening. As the products formed did not adsorb on the
electrode surface, it did not show any peak in CV or LSV. In addition sonication will deter any weak
interaction between the products and the glassy carbon electrode thereby accumulating the product in
the reaction solution itself.
3.2. Spectroscopic studies
3.2.1. UV-Vis analysis
a) Lawsonia inermis
Fig. 5a presents optical absorption band from 400 to 750 nm for 0.1% and 0.5% concentrations of
Lawsonia inermis in ethanol before and after PSEC studies. The major portion of henna leaf extract is
constituted by lawsone (2-hydroxy-1, 4-napthaquinone), resin, tannin, coumarins, gallic acid and
sterols [23, 24]. Lawsonia inermis in ethanol showed four absorbance peaks at 275, 334, 412 and

9
665nm before PSEC perturbation. Peak at 412nm (blue light) was due to chlorophyll-B and 665nm
(red light) was caused by chlorophyll-A [25]. The intensity of the absorbance peak for higher
concentration decreased due to dye degradation after PSEC studies.
b) Lawsone
Khan et al reported that the absorption wavelengths of lawsone in ethanol, involved two bands at 284
and 332 nm [26]. In the current study, lawsone absorbed at 246, 277 and 333nm in ethanol (Fig 5b).
The first absorption peak at 246nm was attributed to the intramolecular hydrogen bonding of
benzoquinone [27]. At 277 nm, the peak was due to the delocalization of electrons from benzonoid
ring to quinonoid ring. The peak at 333 nm accounts for the presence of naphthoquinone in ethanol,
forming hydrogen bonds with the carbonyl groups, as reported in the literature [28-30].
c) PPD
We infer from Fig. 5c that the intensity of longer wavelength peaks decreased after the PSEC
experiments. The peaks were obtained at 244 and 311nm. With increase in PPD concentration the peak
intensity increased without peak shift. Thus one can come to the conclusion that -NH2 group as an
electron releasing entity is involved in the reaction via π electrons conjugation. The band gap energy
of 3.99eV is observed at longer wavelength (311 nm). This is in agreement with the band energy of
natural dye lawsone sharing electronic transition state from HOMO to LUMO. Thus the natural dye
lawsone and synthetic dye PPD has almost similar energy levels in alcoholic medium[31]. Due to this
similarity in their property, PPD is used as colour enhancement agent (adulterant) in henna powder or
henna based hair dye [as discussed in the following section].
d) Band gap energy calculation
When the dye is illuminated with light, it absorbs photon (ℎν) equal or greater in energy than the band
gap and electron gets promoted from the valence band (VB) to the conduction band (CB) [32]. Upon
PSEC perturbation, the dye molecules were excited from the highest occupied molecular orbitals
(HOMOs) to the lowest unoccupied molecular orbital (LUMO) states in ethanol. When electron gets
excited from HOMO to LUMO under PSEC reaction, the excited state of the dye species was further

10
converted into a semi oxidized radical cations (Dye•+). These radical cations are highly reactive and
short lived. It possess the ability to easily absorb UV light via conjugation and react with ethoxy group
resulting in oxidation product. Based on the optical studies of Lawsonia inermis, lawsone and PPD,
the energy corresponding to different electron transitions in the molecule could be identified. Lawsone
exhibit common peak with both henna and PPD. Peak at 3.72eV for lawsone and 3.99eV for PPD
correspond to the hydrogen bonding contributions (Fig 6 and Table 1). In the case of lawsone, peak at
333nm was due to -C=O, -OH hydrogen bonding, whereas for PPD it was due to 311 nm peak caused
by H-N-H (-NH2) hydrogen bonding. The peak wavelength or energy difference was caused by
variation of H-bonding species in both the compounds. The lower wavelength peak at 246nm for
lawsone is attributed to the intramolecular hydrogen bonding leading to conjugation. On the contrary,
peak at 244nm for PPD is due to π electron conjugation. This similarity in the optical energy bands of
lawsone and PPD makes them complimentary to each other in the application of dyes for human usage.
3.2.2 FTIR analysis
a) Lawsonia inermis
The broad peak obtained at 3419cm-1 is allocated to phenolic -OH stretching frequency. The aromatic
C-H stretching band is observed near 3000cm-1. The acidic O-H stretching frequency is identified at
2361 and 2339 cm-1, but after PSEC reaction the acid O-H stretching at 2339 cm-1 vanished. The α-β
unsaturated C=O stretching band is seen at 1633cm-1. The aromatic C=C stretching peak at 1449cm-1
is shifted to 1447 cm-1 after PSEC perturbation. From 1400-1600cm-1, small peaks due to aromatic
C=C stretching were observed. sp3 hybridized C-H bending peak recorded at 1383 and 1316cm-1. The
alkoxy C-O stretching is noticed at 1194 and 1102cm-1 and the aromatic CH2 bending at 780 and
658cm-1. The results show that the aromatic C=C and C-H stretching peaks were observed at low
transmittance. It indicates that the intensity of the peaks after PSEC perturbations decreased without
any change in the peak positions as shown in Fig 7 and Table 2. Thus, we can conclude that upon
PSEC treatment Lawsonia inermis does not undergo any degradation thereby making it a harmless
constituent for dying and tattooing purposes.

11
b) Lawsone
The O-H stretching peak before and after PSEC perturbations appeared at 3175cm-1. The aromatic C-
H stretching were seen before PSEC at 3071 and 3015cm-1 and after PSEC at 3071 and 3010cm-1. The
α-β unsaturated C=O stretching band had been noticed respectively at 1678 and 1641cm-1 before and
after PSEC reactions. The aromatic C=C stretching frequency before and after PSEC reaction is
observed at 1591, 1577 and 1458cm-1. After PSEC reaction, two broad peaks were noticed at 1382
and 1345cm-1, demonstrating the presence of sp3 hybridized C-H bending peak of ethanol attached to
the meta position of the aromatic ring (refer section 3.6). The alkoxy C-O stretching peaks were seen
after PSEC reaction from 1100-1300 cm-1 range. Hence, FTIR confirmed ethoxy group substitution to
the aromatic ring. Aromatic CH2 bending is depicted at the frequency range of 1000-600 cm-1 before
and after PSEC reaction. Thus additional peaks were noticed after PSEC perturbations. Peaks at 1345,
1382, 1282, 1257, 1215 and 1119 cm-1 corresponds to the C-H bending and alkoxy C-O stretching
modes. These additional peaks appear due to degradation products. Thus one may conclude that usage
of lawsone as colouring agent in high concentration would lead to carcinogenic aromatic compounds
as degradation byproducts, leading to skin diseases and other health hazards (Fig 8 and Table 3).
b) PPD
FTIR spectra of reacted PPD possess lower frequency and low transmittance peaks due to dye
degradation caused by PSEC perturbations. In the frequency range 3500-3200 cm-1, NH2 stretching
peak were observed before and after PSEC reaction, the transmittance of the peak decreased due to
degradation causing high frequency red shift. The aromatic -C-H stretching frequency noticed before
reaction at 3008 cm-1 vanished for reacted sample. Thus the aromatic ring was broken after PSEC
reaction and resulted in byproducts (refer section 3.6). In 2000-1600 cm-1 range, two peaks were
identified at 1850 and 1630 cm-1. The para substituent observed before reaction at 1850cm-1 is not
visible after PSEC reaction. At 1630cm-1 N-H bending peak seen before reaction split into two peaks
at 1604 and 1539 cm-1 after reaction. Sharp aromatic C=C stretching peaks were noticed at 1600-1400

12
cm-1 before and after PSEC reaction. Alkene C=C (cyclobutadiene) stretching peak appeared at
1556cm-1 with high transmittance after PSEC reaction. Peak at 1516 cm-1 frequency range indicated
the position of aromatic C-C stretching observed with low transmittance, before reaction and shifted
to 1501cm-1 after reaction. Aromatic C-N stretching frequency seen in the range 1340-1300cm-1 before
reaction shifted to 1350-1250cm-1 indicating aliphatic C-N stretching frequency after reaction. Before
reaction two broad peaks were identified at 831 and 718 cm-1 due to ƥ-disubstituted aromatic C-H
bending, whereas after reaction small peaks for alkene, CH2 bending frequency is noticed. Also, the
shifting of N – H bending band from 798 cm-1 to 813 cm-1 was seen after the PSEC reaction. Thus
these results indicate dye degradation via aromatic ring opening due to UV irradiation, sonication and
electrochemical perturbations.
3.2.3 HPLC analysis
(a) Lawsone
HPLC analysis of lawsone in 50% ethanol show peaks at 0.987 and 1.537 mins at the excitation
wavelength of 280nm. The high intensity peak at 1.537min corresponds to lawsone whereas the low
intensity peak at 0.987 min accounts for the resonance structures (cf. section 3.2.3 b). After PSEC
perturbations there is a shift in the peaks towards higher retention time. The intensity of 1.778 min
peak is 20 times higher than 1.537 min peak (before PSEC). The 1.778 min peak corresponds to
3-ethoxy naphthaquinone (cf. section 3.3a). The peaks at 1.030 and 1.166 mins (couldn’t identify the
peaks in Fig 10) are due to resonance structures of lawsone in its native state. Thus HPLC data
supports the mechanism of degradation of lawsone proposed in section 3.3a. The amount of 3-ethoxy
naphthaquinone present in the solution after PSEC is 85%, thereby demonstrating the near complete
degradation of lawsone to harmful 3-ethoxy naphthaquinone (Fig10).
(b) PPD
HPLC of pure sample of PPD in 50% ethanol showed two characteristic peaks at retention time 4.49
min and 3.21 min at an excitation wavelength of 242nm and is in agreement with the literature [36].
Here peak at 4.49min corresponds to PPD and 3.21 min indicates presence of conjugated PPD[37].

13
After PSEC experiments, the solution containing products were eluted in HPLC at 242nm and in 50%
ethanol. The peaks shifted drastically. Two peaks were noticed at 1.663 and 2.042 retention time. This
lower retention time peaks at 1.663 and 2.042 for the solution after PSEC demonstrates the conversion
of aromatic diamines into aliphatic amino dienes and the absence of peaks at 4.491 and 3.21 indicates
the absence PPD in the product(Fig 11). Thus it is evident that the mechanism proposed in section
3.3b is highly possible for the degradation of PPD in ethanol after PSEC perturbations. The total
quantity of 1-amino cyclobutadiene is 79% and remaining anolyte is in conjugated PPD form with
20% at 242 nm. Thus after PSEC perturbations the degradation is almost complete with 79% of
harmful substituted cyclobutadiene product.
3.2.4. 1H NMR analysis
a) Lawsonia inermis
In Lawsonia inermis before and after PSEC perturbation, two peaks were observed at 2.5 and 3.4ppm,
due to solvent DMSO (Fig 12). In 1H NMR analysis, aromatic and O-H group peaks were observed at
low intensity due to sparing solubility of Lawsonia inermis in DMSO.
b) Lawsone
1H NMR of lawsone in DMSO recorded (Fig 13) three sharp peaks and one less intensity peak. The
peak at 6.14ppm indicated H3 proton in quinonoid structure. 7.75-7.96 ppm denoted the aromatic
protons H5, H6, H7, H8 as doublet and multiplet. 11.64 ppm represented the -OH proton as substituent
to quinonoid ring with weak C-O bond formation. This pure lawsone solution data is compared with
sample after the PSEC perturbation. The chemical shift for lawsone after PSEC reaction being a singlet
peak H3, shifted to 6.16 ppm, aromatic protons were shifted to 7.77-7.98 ppm but the O-H proton
chemical shift vanished. This indicated that after PSEC reaction, proton belonging to O-H group was
terminated from the quinonoid ring in lawsone. Instead ethoxy group protons were observed at 3.5 and
1.5 ppm (Table 5 and Scheme 1).
c) PPD

14
1H NMR of PPD (Fig 14 and Table 6) before and after PSEC perturbations were analyzed. Before
reaction two sharp peaks were noticed, one for -NH2 group at 4.18ppm and another peak for aromatic
protons at 6.39 ppm with all aromatic protons of same type. After PSEC perturbations the final product
gave the 1H NMR data with 6 sharp peaks. Two peaks for solvent, ethoxy group protons were observed
at 1.5 and 3.5 ppm, remaining 4 sharp peaks had protons at different chemical environment due to
intramolecular hydrogen shifting in the aromatic ring. The resonance structures of PPD are depicted
in Scheme 2.
3.2.5 13C NMR analysis
(a) Lawsone:
The chemical shift of lawsone (Fig 15 a) C1 is about 138 ppm, C3 is observed at 152 ppm which is on
higher field. On the other hand the chemical shift of C2 is observed on lower field (114 ppm). The
chemical shifts of C3 & C4 are noticed at 151 and 145 ppm. The considerable lower field shift for C4
is reasonable due to C=O. The chemical shifts of C5 to C10 were observed at 114, 140, 141, 128, 129,
13
142 ppm. These data agree well with the C NMR spectra of lawsone reported in the literature[39].
After PSEC perturbations, the samples were analysed for 13C NMR, the chemical shifts were almost
identical to lawsone, except for ethoxy carbon peaks at lower field of 116ppm, C7 to C10 carbons
grouping at 128ppm. This grouping of C7-C10 carbons indicate identical chemical environment for
the product obtained. C2 at 152ppm, showing the shift of hydroxyl group attached to C2 into carbonyl
group. C3 chemical shift is at 114ppm whereas the chemical shift of remaining carbons (C1, C3-C6)
appears at the same position as lawsone (Fig 15b). Thus from these chemical shifts, we can infer that
the product formed is identical in structure to lawsone except for an carbonyl group at C2 and ethoxy
13
group at C3. Thus C NMR analysis confirms the product obtained after PSEC perturbation as 3-
ethoxy naphthaquinone.
(b) PPD:
13
Pure PPD standard show two environments in C NMR. One peak at 138 ppm contributed by -
13
carbons to which amino groups are attached. The o- and m- position carbons appear at 117ppm. C

15
NMR spectra of PPD in 50% ethanol after PSEC perturbations displayed peaks corresponding to
different carbon environments in different positions. Here two sharper and small peaks are identified,
one at 115ppm and the other at 138ppm. These small intensity peaks are attributed to the unreacted
conjugated PPD present in the solution after PSEC perturbations. The sharp peak at 40ppm, when
analyzed carefully is found to split into septet (40.3, 39.9, 39.7, 39.5, 39.2, 39, 38 ppm). These
peaks correspond to different carbon environments in 1-amino cyclobutadiene. The amino group
substituted carbon (C1) appeared at 40ppm, the neighbouring carbon atoms (C2 and C4) are affected
by C3 carbon as well as C1 carbon leading to a triplet, C3 carbon inturn is affected by C1, C2 and C4
environment leading to triplet. Thus a singlet peak at 40ppm is split further with very little chemical
shift forming a septet. Thus 13C NMR analysis supports the formation of 1-amino cyclobutadiene as
the predominant product from PPD upon PSEC perturbations (cf. Fig 16).
3.3. Degradation mechanism based on PSEC perturbations and spectral analysis
a) Degradation pathway for Lawsone:
.
The possible degradation mechanism for lawsone is obtained based on HPLC, UV-Vis, FTIR and 1H
NMR and 13C NMR spectroscopic analysis as follows:
Step 1
Step 2

16
Lawsone being a photo sensitive dye upon UV irradiation, hydrogen radical elimination occurred as
1
supported by H NMR analysis data. As the radical species is short lived intermediate and get
extensively involved in the resonance structure (step 3), peaks corresponding to the radical is not
observed in UV-Vis spectra.
Step 3
Lawsone undergo electron shifting due to the stabilization of oxygen radical by taking the π electrons
in the naphthoquinone group via π electron conjugation.
Step 4
Naphthoquinone radical reacted with ethoxy radical (chromophore) and form new bond.
Hence the degradation mechanism is shown as given below.
The above degradation mechanism is deduced and supported by HPLC, UV-Vis, FTIR, 1H NMR and
13C NMR spectroscopic analysis. In UV analysis the peak intensity was increased due to chromophore
attachment to lawsone by the removal of hydrogen radical. In FTIR ethoxy group attached to the
naphthoquinone after PSEC perturbation showed peaks at 1345, 1382, 1282, 1257, 1215 and 1119 cm-
1
1. H NMR results showed the alcohol peak at 3.5 ppm and hydroxyl peak at 11.6 ppm before the
PSEC reaction disappeared after PSEC perturbations. HPLC accounted for the formation of 3-ethoxy

17
naphthaquinone as predominant product via retention time peak at 1.778 min. 13C NMR analysis
proves this product formation via the chemical shift of C2 and C3 carbon dissimilar to lawsone
(presence of C2 carbonyl and C3 ethoxy carbons).
b) Degradation pathway for PPD
Step 1
Step 2
Step-2 shows that the π electrons were excited due to PSEC treatment. So PPD resulted in radical
formation.
Step 3
In step-3 the radical form of PPD reacted with ethoxy radical to form a new compound. This ethoxy
group attachment to aromatic ring at meta position was supported by 1H NMR data via the splitting
of aromatic C-H peaks due to change in the environment.

18
Step 4
In step-4 formation of a bridged compound due to PSEC perturbation is noticed.
Step 5
In step-5 upon PSEC perturbation, 1,2 diamino cyclobutadiene and ethoxy ethylene were formed as
evident from FTIR results at 1234 cm-1 due to aliphatic C-N-C bending. Ethoxy group is observed
in 1H NMR analysis at 3.5ppm.
Step 6
In step-6 another mole of PPD reacted with 1,2 diamino-cyclobutadiene to form an addition compound
with removal of ammonia. The product formed is supported by 1H NMR analysis at 4.1, 4.9(-NH2),
7.5-7.9ppm(-NH-) and the path followed is similar to Diels Alder reaction.
Step 7

19
Upon further perturbation via PSEC experiments, in step-7 the bond breaking occurred resulting in
resonance structure of PPD and 1-amino cyclobutadiene.
The overall mechanism is displayed below with the support of spectroscopic analysis.
1
The mechanism was deduced based on FTIR and H NMR analysis. In FTIR analysis, after PSEC
perturbations the peak shift was observed from aromatic to aliphatic C-N-C bending from 1262 to
1
1234 cm-1, in H NMR analysis new peaks were identified at low intensity due to formation of new
compound and aromatic peaks were also observed at 4.9 ppm, 7.5-7.9 ppm, and 5.7-6.7 ppm. The
new peaks in 1H NMR were due to formation of products such as ethoxy group, -NH group and -NH2
group after PSEC reaction. HPLC analysis of PPD after PSEC perturbations revealed two peaks at
1.663 and 2.042 retention time. This lower retention time peaks at 1.663 and 2.042 for the solution
after PSEC demonstrates the conversion of aromatic diamines into aliphatic amino dienes and the
absence of peaks at 4.491 and 3.21 indicates the absence PPD in the product. 13C NMR analysis peaks
correspond to different carbon environments in 1-amino cyclobutadiene. The amino group substituted
carbon (C1) appeared at 40ppm, the neighbouring carbon atoms (C2 and C4) are affected by C3 carbon
as well as C1 carbon leading to a triplet, C3 carbon in turn is affected by C1, C2 and C4 environment
leading to triplet. Thus a singlet peak at 40ppm is split further with very little chemical shift forming
a septet. Thus 13C NMR analysis supports the formation of 1-amino cyclobutadiene as the predominant
product from PPD upon PSEC perturbations.

20
Summary
PSEC perturbation studies of Lawsonia inermis (natural dye, henna), lawsone and PPD were carried
out in ethanol. The PSEC studies demonstrated that (i) lawsone, a major constituent of Lawsonia
inermis donot undergo degradation upon exposure to UV – irradiation, sonication and electrochemical
perturbation and hence the natural dye can stay longer on the skin or fabric without any harmful effects,
(ii) commercial lawsone added as colouring agent to henna by dye and tattoo industry responded as
an electroactive compound undergoing oxidation upon PSEC perturbation resulting in the formation
of ethoxy (at meta position) substituted naphthaquinone as demonstrated by the spectroscopic analysis
and (iii) PPD, an artificial dye added as adulterant to henna to enhance its colouring property resulted
in the formation of amino substituted cyclobutadiene upon PSEC perturbations. Thus from the present
findings, formation of carcinogenic [40,41] ethoxy substituted naphthaquinones and amino substituted
cyclobutadiene by lawsone and PPD respectively demonstrated the harmfulness in utilizing these
artificial dyes as colour enhancing agent to henna by dye and tattoo industries.
Acknowledgements: The authors acknowledge the UV-Vis experimental facility provided by Prof.
B. Neppolian, SRM Research Institute. The authors also acknowledge the experimental facilities by
Department of Science and Technology – Fund for Improvement of Science and Technology
Infrastructure (DST-FIST, Govt of India) at Department of Physics and Nanotechnology and
Department of Chemistry, SRM Institute of Science and Technology, Kattankulathur, India.
Authors gratefully acknowledge the valuable comments and suggestions by the reviewers that
substantially improved the manuscript.

21
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Fig.1. Cyclic Voltammograms in ethanol under UV irradiation with sonication (solid lines) and after
3 hrs of UV irradiation and sonication (dotted lines) of (a) 0.1% Lawsonia inermis (b) ) 0.5%
Lawsonia inermis (c) 0.1% Lawsone (d) 0.5% Lawsone (e) 0.1% PPD and (f) 0.5% PPD.
Fig.2 (a) D1/2 vs ν of 0.1% and 0.5% lawsone in ethanol and (b) ip vs ν of 0.1% and 0.5%
lawsone in ethanol

26
Fig.3. Linear sweep voltammetric analysis in ethanol of (a) 0.1% Lawsone (b) 0.5% Lawsone (c)
0.1% PPD (d) 0.5% PPD

27
Fig.4 (a) D1/2 Vs ν of 0.1% and 0.5% lawsone in ethanol and (b) i Vs ν1/2 of 0.1% and 0.5% lawsone
in ethanol

28

29
Fig 5. UV-Vis spectrum in ethanol 0.1% and 0.5% concentrations before (solid lines) and after PSEC
perturbations (dotted lines) of (a) Lawsonia inermis (b) Lawsone (c) PPD.