Impact of Biofield Treatment on Spectroscopic and Physicochemical Properties of p-Nitroaniline

Para nitroaniline ( p -Nitroaniline) is an organic compound, used as an intermediate in the synthesis of pharmaceuticals drugs, gasoline and dyes. The present study was attempted to investigate the influence of biofield treatment on p -nitroaniline. The study was performed in two groups i.e., control and treatment. The treatment group was subjected to Mr. Trivedi’s biofield treatment. The control and treated samples of p -nitroaniline were characterized using Fourier transform infrared (FT-IR) spectroscopy, Ultraviolet-visible (UV-Vis) spectroscopy, high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and differential scanning calorimetry (DSC). FT-IR spectral analysis result suggested the alteration in wavenumber of some groups with respect to control. For instance, the C=C and C-C stretching were observed at 1570 cm -1 and 1430 cm -1 , respectively in control sample that were shifted to 1585 cm -1 and 1445 cm -1 , respectively after biofield treatment. UV spectral analysis revealed the similar pattern of absorbance maxima (λ max ) in both control and treated samples. HPLC data showed an alteration in the retention time of p -nitroaniline peak in treated sample (3.25 min) with respect to control (2.75 min). GC-MS results showed a significant change in the isotopic abundance (δ) of 13 C and 18 O in treated sample as compared to control. DSC data showed that latent heat of fusion (∆H) of treated p-nitroaniline was substantially decreased by 10.66% as compared to control. However, the melting point remained same in both control and treated sample of p-nitroaniline. Overall, results obtained from different analytical techniques such as FT-IR, HPLC, GC-MS, and DSC suggested that biofield treatment has significant impact on spectral, physical and thermal properties of p-nitroaniline with respect to control sample.


Introduction
Aromatic amines are very important in biology and chemical industry.Particularly aniline and its derivatives are being used as antioxidants, and in production of dyes and pesticides [1,2].Additionally, the aromatic amines are also used as intermediate in the synthesis of several pharmaceutical drugs including acetaminophen, nadifloxacin, difloxacin, sarafloxacin, flumequin, cisapride, bicalutamide, brequinar, sorbinil etc. [3,4].Some of para (p) substituted anilines have local anesthetic property, wherein amino group plays an important role in the interaction with respective receptor.p-Nitroaniline or 4-nitroaniline (C 6 H 6 N 2 O 2 ) is one of the important compounds of this class [2].It is a bright yellow powder with a faint ammonia-like odor.It is mainly used as an intermediate for the synthesis and preparation of several antioxidants, antiseptic agents, medicines for poultry and other pharmaceutical products [5].Diazo product of this compound can be used for the production of azo dye in the textile industry.Some other chemical intermediates like p-phenylenediamine, 2-chlor-4-nitroaniline, 2,6-dichloro-4-nitroanilen etc. are also prepared using p-nitroaniline as a starting material [6].The p-nitroaniline is widely used as an intermediate in various chemical reactions; therefore, its rate of reaction or reactivity is crucial.Previously published report suggested that reaction kinetics of any chemical reaction depend on the physical and thermal properties of intermediate compounds i.e., latent heat of fusion, and vaporization temperature etc. [7,8].Hence, it is advantageous to find out an alternate approach, which could alter the physical, thermal and spectral properties of chemical compounds.Recently, several studies have been reported on biofield treatment to alter the spectral properties of various pharmaceutical drugs like metronidazole, tinidazole, paracetamol, and piroxicam; and physical, and structural properties of various metals i.e., tin, lead etc. [9][10][11].The relation between mass-energy (E = mc 2 ) was described by Einstein [12].Furthermore, the energy exists in various forms and there are several ways to transfer the energy from one place to another such as electromagnetic waves, electrochemical, electrical and thermal etc.Similarly, the human nervous system consists of neurons, which have the ability to transmit information and energy in the form of electrical signals [13,14].Thus, the human has the ability of energy harness from the environment or Universe and transmit this energy into any object (living or nonliving) on the Globe.The object(s) always receive the energy and responding into useful way, this process is known as biofield treatment or healing [15].The National Center for Complementary and Alternative Medicine (NCCAM) considered this biofield treatment (therapy) in subcategory of energy therapies [16].Mr. Trivedi's unique biofield energy is also known as biofield treatment (The Trivedi Effect → ).This effect is known to change the physicochemical, thermal and structural properties of metals [11,17] and ceramics [18].Considerable changes in overall growth and yield of medicinal and agricultural plants were also reported after biofield treatment [19].Further, biofield treatment has substantially altered the antimicrobial susceptibility, biochemical reactions pattern, and biotype number of several pathogenic microbes [20,21].Conceiving the impact of biofield treatment on various living and nonliving things, the study was aimed to evaluate the impact of biofield treatment on spectral and physicochemical properties of p-nitroaniline.The effects were analyzed in both control and treated p-nitroaniline samples using Fourier transform infrared (FT-IR) spectroscopic, ultraviolet-visible (UV-Vis) spectroscopy, high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and differential scanning calorimetry (DSC).

Study design
The p-nitroaniline was procured from Sigma-Aldrich, Mumbai, India.The study was performed in two groups of p-nitroaniline i.e., control and treatment.The control sample was named as untreated, and treatment sample was handed over in sealed pack to Mr. Trivedi for biofield treatment under laboratory condition.Mr. Trivedi provided this treatment through his energy transmission process to the treatment group without touching the sample [10].The control and treated samples of p-nitroaniline (Figure 1) were evaluated using various analytical techniques viz.FT-IR, UV-Vis, HPLC, GC-MS, and DSC.

FT-IR spectroscopic characterization
FT-IR spectra of control and treated samples of p-nitroaniline were recorded on Shimadzu's Fourier transform infrared spectrometer (Japan) with frequency range of 4000-500 cm -1 .The analysis was carried out to evaluate the impact of biofield treatment at atomic level like dipole moment, force constant and bond strength in chemical structure [22].

UV-Vis spectroscopic analysis
UV spectra of control and treated samples of p-nitroaniline were recorded on Shimadzu UV-2400 PC series spectrophotometer with 1 cm quartz cell and a slit width of 2.0 nm.The analysis was carried out using wavelength in the range of 200-400 nm.The UV spectral analysis was performed to determine the effect of biofield treatment on the energy gap of bonding and nonbonding transition of electrons [22].

HPLC analysis
HPLC analysis of control and treated samples of p-nitroaniline were carried out on KNAUER's Smartline series of HPLC (Berlin, Germany) with Smartline pump 1000 and UV 2600 detector.The separation was carried out on Eurospher 100-C 18 column (250 × 4 mm) using methanol as a mobile phase.The analysis was carried out to evaluate the impact of biofield treatment on the purity and retention time (τ Ρ ) of p-nitroaniline with respect to control.Alteration in retention time of analyte in identical HPLC conditions refers to changes in the polarity of analyte.

Gas chromatography-mass spectroscopy (GC-MS) analysis
The p-nitroaniline sample was divided into four groups: one was set as control and another three were coded as treatment i.e., T1, T2, and T3.The GC-MS analysis of control and treatment samples of p-nitroaniline was performed on Perkin Elmer/auto system XL with Turbo mass and electron ionization mode, USA.Detection limit was set up to 1 pico gram, and mass range was set to 10-650 amu.The isotopic ratio 13 C/ 12 C was expressed by its deviation in treated p-nitroaniline sample with respect to control.The isotopic abundances of 13 C and 18 O were computed on a delta scale per thousand.The values of δ 13 C and δ 18 O of treated samples were calculated using following equation.Where, R Treated and R Control are the ratio of intensity at m/z=139/ m/ z=138 for δ 13 C, and m/z=140/ m/z=138 for δ 18 O in mass spectra of treated and control samples respectively.

Differential scanning calorimetry (DSC) analysis
The control and treatment samples of p-nitroaniline were analyzed by using a Pyris-6 Perkin Elmer DSC on a heating rate of 10°C/min under air atmosphere and air was flushed at a flow rate of 5 mL/min.Percent change in latent heat of fusion was calculated using following equations: Where, ΔH Control and ΔH Treated are the latent heat of fusion of control and treated samples, respectively.

FT-IR spectroscopic analysis
Vibrational spectral assignment was performed on the recorded FT-IR spectra (Figure 2a and 2b) based on theoretically predicted wavenumber and presented in Table 1.The p-nitroaniline has one NH 2 group therefore one symmetric and one asymmetric N-H stretching vibrations are expected that were assigned to peaks appeared in the region of 3350-3478 cm -1 in control and 3362-3482 cm -1 in treated sample.It showed the upstream shifting of wavenumber of N-H bond in treated sample with respect of control.The frequency (ν) or wavenumber of stretching vibrational peak depends on two factors i.e., force constant (k) and reduced mass (μ), which can be explained by following equation [23].
If μ is constant, then the frequency is directly proportional to the force constant; therefore, increase or decrease in frequency of any bond indicates a corresponding increase and decrease in force constant [22].Based on this, it is speculated that the increased in wavenumber of N-H bond after biofield treatment might be observed due to increased force constant of respective bond as compared to control.In addition to N-H stretching, the NH 2 group also has scissoring, rocking wagging and torsion modes of fundamental vibrations [2].The NH 2 scissoring vibrational mode was assigned to peak appeared at 1628 cm -1 in control and 1630 cm -1 in treated sample of p-nitroaniline.Theoretically, rest of NH 2 group vibrations should appeared below 500 cm -1 that were not recorded in the spectra (Figure 2).The asymmetric vibration of NO 2 group was assigned to peaks observed at 1507 cm -1 and 1506 cm -1 in control and treated sample, respectively.Likely the symmetric vibration of NO 2 group was assigned to peak observed at 1345 cm -1 and 1344 cm -1 , in control and treated sample respectively.The result showed that both the N-H bending and NO 2 asymmetric-symmetric vibrations in control and treated samples were observed in the similar frequency region.However, the FT-IR spectrum of treated sample showed the enhanced intensity of NO 2 symmetric peak with respect to control.The existing literature on FT-IR suggested that intensity of vibrational peak of particular bond depends on ratio of change in dipole moment (∂µ) to change in bond distance (∂r) i.e., the intensity is directly proportional to change in dipole moment and inversely proportional to change in bond distance [24].Based on this, it is speculated that the increase in intensity of NO 2 symmetric stretching in treated sample might be due to alteration in the ratio of ∂µ/∂r as compared to control.The C=C (aromatic) stretching was attributed to vibrational peak appeared at 1570 cm -1 and 1585 cm -1 in control and treated sample, respectively.Likewise, the C-C stretching was assigned to peak appeared at 1430 cm -1 in control and 1445 cm -1 in treated sample.The result showed that both C=C and C-C stretching vibrations were shifted to higher wavenumber in treated sample with respect to control.

Insights in Analytical Electrochemistry ISSN 2470-9867
This might be due to induced force constant and bond strength of respective bonds in treated sample with respect to control.The C-N stretching vibrations mode were assigned to peaks observed at 1244-1283 cm -1 in control and 1300-1335 cm -1 in treated sample.Further the C-H in plane bendings were assigned to peak at 1103-1174 cm -1 and 1113-1183 cm -1 in control and treated sample respectively.The increase in wavenumber of C-N stretching and C-H in plane bending after biofield treatment depicted an increase in force constant and torsional force, respectively due to influence of biofield treatment.The out of plane C-H bending peaks were assigned at 664-779 and 633-842 cm -1 in control and treated sample, respectively that also showed the biofield induced alteration in the wavenumber as compared to control.All together, the FT-IR data showed a significant change in the wavenumber and intensity of some bonds in treated sample with respect to control.This could be due alteration in force constant, dipole moment and bond length of respective bonds as compared to control.

UV-Vis spectroscopy
UV spectral analysis of control p-nitroaniline showed absorbance maxima (λ max ) at 226.4 and 359.6 nm, which was also supported by the literature data [25].After biofield treatment, the absorbance maxima (λ max ) values were slightly shifted at 228.0 nm and 362.0 nm in treated sample.The result showed the slight bathochromic shift in both the absorption peaks (λ max ) in treated sample.The compound can absorbs UV light due to the presence of either or both conjugated pi (π) -bonding systems (π-π* transition) and nonbonding electron system (n-π* transition).There is certain energy gap between π-π* and n-π* orbitals.When this energy gap altered, the wavelength (λ max ) was also altered respectively [22].However, the UV study showed the similar pattern of absorbance maxima in both control and treated samples of p-nitroaniline.Based on this, it is speculated that, biofield treatment did not alter the energy gap between π-π* and n-π* transition in p-nitroaniline.

HPLC analysis
The HPLC chromatogram of control and treated p-nitroaniline are shown in Figure 3.The HPLC chromatogram of control p-nitroaniline exhibited the corresponding peak at 2.75 min (Figure 3a), while the treated p-nitroaniline peak was observed at 3.25 min (Figure 3b).The position of peak corresponding to analyte in the HPLC chromatogram depends on its polarity.In reverse phase HPLC, if the retention time increases in the same experimental condition, it suggests the increases in nonpolar property of respective analyte.Similarly, the decrease in retention time suggests an increase in polar property in the corresponding analyte sample [26].The FT-IR spectral analysis suggested that biofield treatment has induced alteration in dipole moment of treated p-nitroaniline.Dipole moment can be referred to polarity in the molecule, therefore alteration in dipole moment can lead to alteration in the polarity, which could be seen in the HPLC analysis (retention time) result.Herein, we also observed a significant increase in the retention time of p-nitroaniline in treated sample as compared to control, which suggested that after biofield treatment some nonpolar property might be induced in p-nitroaniline with respect to control.

GC-MS analysis
The GC-MS spectra of control and treated (T1, T2, and T3) p-nitroaniline are shown in Figure 4a and 4b, and the peak intensity of most probable isotopes are illustrated in Table 2.
The GC-MS spectra of control and treated samples showed the primary molecule (PM, p-nitroaniline) peak at m/z 138 suggest the molecular weight of p-nitroaniline.In addition the peaks at m/z 139 and m/z 140 were assigned to isotopic abundance peaks due to PM +1 and PM +2 isotopes of p-nitroaniline [27].It is well known that 13 C and 18 O isotope are more stable and exist in higher percentage than the 3 H, 2 H, 15 N, and 17 O isotopes.Therefore, it is presumed that isotopic abundance ratio of PM +1 is mainly due to 13 C and PM +2 is may be due to 18 O isotope in p-nitroaniline.Based on this it is speculated that the peak at m/z 138 is mainly due to 12  O 1 .The GC-MS analysis result (Table 2) suggested that the isotopic abundance of 13 C (δ 13 C) was changed as -23.33, -63.31, and -30.15‰ in T1, T2, and T3, respectively; and the isotopic abundance of 18 O (δ 18 O) was changed as -8.22,-56.54, and -44.43‰ in T1, T2, and T3, respectively as compared to control.The result depicted that in all the treated sample the 13 C and 18 O were transformed into 12 C and 16 O, respectively by releasing the neutron(s) (one from 13 C and two from 18 O).This inter-conversion of 13 C -12 C, and 18 O - 16 O might possible if a nuclear level reaction occurred due to influence of biofield treatment.Based on this, it is postulated that biofield treatment possibly induced the nuclear level reactions in p-nitroaniline, which may lead to convert the 13 C and 18 O into 12 C and 16 O, respectively.

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HPLC chromatogram showed an alteration in the retention time of p-nitroaniline peak with respect to control, which is possibly due to alteration in the polarity of treated sample as compared to the control.The GC-MS analysis showed that isotopic abundance of 13 C (δ 13 C) was altered about -23.33, -63.31, and 30.15‰ in T1, T2, and T3, respectively; whereas.The δ 18 O was altered about -8.22, -56.54, and -44.43‰ in T1, T2, and T3, respectively.This might be occurred due to the biofield energy mediated changes at nuclear level reaction in the treated sample.The DSC analysis showed the considerable change (10.66%) in latent heat of fusion in treated sample as compared to control.It may be due to transformation of p-nitroaniline to lower energy state after biofield treatment.Altogether, the FT-IR, HPLC, GC-MS and DSC results suggest that biofield treatment has significant effect on structural, physical and thermal properties of biofield treated p-nitroaniline with respect to control sample.This might be corroborated to possible changes in chemical and thermal stability of treated p-nitroaniline.

DSC study
DSC was used to analyze the melting temperature and latent heat of fusion (ΔH) of control and treated p-nitroaniline.The DSC thermogram of control and treated p-nitroaniline are shown in Figure 5.In a solid content, substantial amount of interaction force exists in atomic bonds that hold the atoms at their positions.ΔH can be defined as the energy required to overcome this interaction force to change the phase from solid to liquid.Hence, the energy provided during phase change i.e., ΔH is stored as potential energy of atoms.However, melting point is related to kinetic energy of the atoms [28].The DSC thermogram showed the melting temperature at 149.31°C for control and 149.18°C for treated sample, which revealed the similar melting point in control and treated sample of p-nitroaniline.The melting temperature of p-nitroaniline is well supported by literature data [29].The DSC thermogram showed the 142.92 J/g latent heat of fusion (∆H) in treated sample and 159.98 J/g in control sample.The result showed decrease in latent heat of fusion in treated p-nitroaniline by 10.66% in comparison with control.It may be due to conversion of p-nitroaniline to lower energy state (lower potential energy) after biofield treatment.Previously, our group has been reported that biofield treatment has shown alteration in latent heat of fusion of lead and tin powders [11].Therefore, it is assumed that biofield treatment might alter the potential energy of treated p-nitroaniline that may lead to change the latent heat of fusion.

Conclusion
FT-IR spectrum of treated p-nitroaniline showed the alteration in wavenumber of IR peaks assigned to N-H, C=C, C-C stretching, and C-H deformation vibrations.This is might be occurred due to alteration in the dipole moment and force constant of the respective bond in treated sample as compared to the control.

Figure 1
Figure 1 Chemical structure of p-nitroaniline.

Figure 2
Figure 2 FT-IR spectra of p-nitroaniline (a) control and (b) treated.

Figure 3
Figure 3 HPLC chromatogram of p-nitroaniline (a) control and (b) treated.

Table 1
FT-IR vibrational peaks observed in p-nitroaniline.