Ferrous sulphate heptahydrate or iron (II) sulphate heptahydrate (>99%) was purchased from Sigma-Aldrich, India. All other chemicals used during the experiments were of analytical grade available in India.

The test compound i.e., iron sulphate was taken and separated into two different parts. In one part, no treatment was given and the sample was considered as control iron sulphate. Besides, in the other part, the Energy of Consciousness Healing Treatment was provided by the renowned Biofield Energy Healer, Mr. Mahendra Kumar Trivedi (USA), and the sample was named as the Biofield Energy Treated iron sulphate. In the process of Biofield Energy Treatment, the sample was placed under the standard laboratory conditions and the Healer remotely provided the Trivedi Effect^® - Energy of Consciousness Healing Treatment to the sample for 3 minutes through the Unique Energy Transmission process. Consequently, the control sample was subjected under the similar laboratory conditions to “sham” healer, who did not have any knowledge about the Biofield Energy Treatment. Later on, both the control and Biofield Energy Treated samples were kept in similar sealed conditions and characterized by using various analytical techniques such as PXRD, PSA, UV-Vis, FT-IR, TGA/DTG, and DSC techniques for analyzing the impact of Biofield Energy Treatment on the iron sulphate sample.

The particle size analysis involves wet method, which is done using Malvern Mastersizer 3000, UK. The instrument has a detection range between 0.01 µm to 3000 µm 41, and the method involves the filling of sample unit (Hydro MV) with light liquid paraffin oil, which acts as dispersant medium. Further, it was stirred at 2500 rpm. The refractive index values for dispersant medium and samples were 0.0 and 1.47, respectively. The measurement was taken twice after reaching obscuration in between 10% and 20%, and the average of both the measurements were done consequently. The PS analysis provides data in the form of d₁₀ μm, d₅₀ μm, and d₉₀ μm, representing the particle diameter corresponding to 10%_, 50%_, and 90% of the cumulative distribution. D(4, 3) μm represents the average mass-volume diameter and SSA is the specific surface area (m²/Kg). The calculations were done by using software Mastersizer V3.50.

The percent change in particle size (d) for d₁₀,d₅₀,d₉₀ and D(4,3) was calculated using following equation 3:

...(3)

Where, d_Controland d_Treatedare the particle size (μm) for at below 10% level (d₁₀)_, 50% level (d₅₀)_, and 90% level (d₉₀) of the control and Biofield Energy Treated samples, respectively.

Percent change in surface area (S) was calculated using following equation 4:

...(4)

Where, S_Control and S_Treated are the surface area of the control and Biofield Energy treated iron (II)sulphate, respectively.

The UV-Vis spectral analysis of the control and Biofield Energy Treated iron sulphate samples was carried out using Shimadzu UV-2400PC SERIES with UV Probe (Shimadzu, JAPAN). The spectrum was recorded in the wavelength range of 190-800 nm using 1 cm quartz cell having a slit width of 0.5 nm. The absorbance spectra (in the range of 0.2 to 0.9) and wavelength of maximum absorbance (λ_max) were recorded.

FT-IR spectroscopy of iron (II) sulphate was performed on Spectrum ES Fourier transform infrared spectrometer (Perkin Elmer, USA) by using pressed KBr disk technique with the frequency array of 400-4000 cm^-1. The technique uses ~2 mg of the control sample and about 300 mg of KBr as the diluent to form the pressed disk followed by running the sample in the spectrometer. The same procedure was used for the Biofield Energy Treated sample.

TGA/DTG thermograms of control and Biofield Energy Treated iron sulphate samples were obtained using TGA Q500 themoanalyzer apparatus, USA under dynamic nitrogen atmosphere (50 mL/min). It involves the heating rate of 10 ºC/min from 25 °C to 800 °C and uses platinum crucible 42. In TGA analysis, the weight loss in gram as well as percent loss for each step was recorded with respect to the initial weight of the sample. Later on, in DTG analysis, the onset, endset, peak temperature and integral area for each peak was recorded. The percent change in weight loss (W) was calculated using following equation 5:

...(5)

Where, W_Control and W_Treated are the weight loss of the control and Biofield Energy Treated samples, respectively.

Also, the percent change in maximum thermal degradation temperature (T_max) (M) was calculated using following equation 6:

...(6)

Where, M_Control and M_Treated are the T_max values of the control and Biofield Energy Treated samples, respectively.

The DSC analysis of the samples was performed using DSC Q2000 differential scanning calorimeter, USA under the dynamic nitrogen atmosphere with flow rate of 50 mL/min. For analysis, 2-4 mg sample was weighed and sealed in aluminium pans. Further, it was equilibrated at 30°C and heated up to 450ºC at the heating rate of 10º C/min under Nitrogen gas as purge atmosphere 42. The value for onset, end set, peak temperature, peak height (mJ or mW), peak area, and change in heat (J/g) for each peak was recorded. Later on, the percent change in melting temperature (T) of the control and Biofield Energy Treated samples was calculated using following equation 7:

(7)

Where, T_Control and T_Treated are the melting temperature of the control and Biofield Energy Treated iron sulphate samples, respectively.

Also, the percent change in the latent heat of fusion (ΔH) was calculated using following equation 8:

(8)

Where, ΔH_Controland ΔH_Treatedare the latent heat of fusion of the control and treated iron sulphate, respectively.

The PXRD diffractograms of the control and Biofield Energy Treated samples of iron sulphate are given in Figure 1. The diffractograms showed very sharp peaks, which represents the crystalline nature of both the samples. Also, the PXRD data is presented in Table 1, which involves the analysis of Bragg angle (2θ), relative peak intensity (%), and crystallite size (G) for the control and Biofield Energy Treated iron sulphate from the diffractograms. The crystallite size was calculated using Scherer equation 43.

The highest intense peak (100% relative intensity) in the control sample was observed at Bragg’s angle (2θ) equal to 18.3° (Table 1, entry 4), while it was observed in the Biofield Energy Treated sample at 2θ equal to 18.1° (Table 1, entry 4). The Bragg’s angle (2θ) of the remaining diffraction peaks in both the samples remained almost same, but the relative intensities of the peaks of the Biofield Energy Treated sample were found to be altered compared to the control sample. The relative intensities of the PXRD peaks at 2q equal to 11.1°, 13.1°, 16.2°, 22.1°, 27.4°, 32.8°, 34.2°, 36.9°, and 54.2° (Table 1, entry 1-3, 5, 7-10, and 12) in the Biofield Energy Treated sample were significantly increased in the range from 24.63% to 301.40%, compared to the control sample. Subsequently, the relative intensities of the PXRD peaks at 2q equal to 23.6° and 47.1° (Table 1, entry 6 and 11) in the Biofield Energy Treated sample were significantly decreased by 39.49% and 35.12%, respectively, as compared to the control sample. On the other hand, the crystallite size values of the Biofield Energy Treated sample at 2q equal to 11.1° and 22.1° (Table 1, entry 1 and 5) was significantly reduced by 15.40% and 8.34%, respectively with respect to the control sample. Subsequently, the crystallite size values of the Biofield Energy Treated samples at 2q equal to 13.1°, 18.3/18.1°, 23.6°, 27.4°, 32.8°, 34.2°, 36.9°, and 47.1° (Table 1, entry 2, 4, and 6-11) were significantly increased from 7.71% to 33.36% in the Biofield Energy Treated sample in comparison to the control sample. However, the crystallite size values of the control and Biofield Energy Treated iron sulphate at position 2q equal to 16.2° and 54.2° (Table 1, entry 3 and 12) remained unchanged. Also, the average crystallite size of the Biofield Energy Treated iron sulphate was significantly increased by 4.98%, compared with the control sample.

The significant variations in the crystallite size and relative intensities of the Biofield Energy Treated iron sulphate indicated the modification of the crystal morphology of the sample compared with the control sample. Several studies reported the impact of Biofield Energy Treatment in producing a new crystalline polymorph of the compound by altering the crystal morphology through the changes in the relative intensities and crystallite size of the characteristic diffraction peaks 353637. Thus, the Trivedi Effect^® - Energy of Consciousness Healing Treatment probably led to produce a new polymorphic form by transferring the energy into iron sulphate sample. The polymorphism plays significant role in the performance of drug, such as its bioavailability, efficacy, and toxicity, due to the alterations in their physicochemical and thermodynamic properties from the original form 4546. Thus, the Biofield Energy Treatment could be considered as a useful method for the production of new polymorph of iron sulphate with improved drug performance.

The UV-visible spectra of the control and treated iron sulphate samples are shown in the Figure 2.

The UV spectrum of the control and biofield energy treated samples showed the maximum absorbances at 190 nm (λ_max). The peak at 190 nm was showed a minor shift of absorbance maxima from 0.6367 in the control sample to 0.6129 in the Biofield Energy Treated sample. The analysis represents that there might not be any significant change in the electronic transitions between highest occupied molecular orbital and lowest unoccupied molecular orbital, induced by the Biofield Energy Treatment.

The FT-IR spectra of the control and treated samples of iron sulphate are shown in Figure 3. The FT-IR spectra of both control and Biofield Energy Treated sample showed the clear stretching and bending peak that are matched well with the available literature 5152. The transitions include the stretching and bending modes of O―H and H―O―H bonds, and stretching vibrations of the SO₄ group. According to the literature, the stretching O―H vibrations (νOH) takes place at 2900-3660 cm^-1 and M―O―H bending vibrations (δOH) at 900-1170 cm^-153. In these spectra, the broad peaks were observed near 3342 and 3378 cm^-1 in the functional group area of the control and Biofield Energy Treated samples, respectively, due to the presence of O―H vibrations of water molecules. The spectra showed S=O stretching at 1094 and 1095 cm^-1 for the control and Biofield Energy Treated samples, respectively. The FT-IR spectra of both samples did not display any alteration in the vibrational frequencies, which represents that there was no alteration in the structural properties associated with a change in dipole moment in the molecule in the Biofield Energy Treated sample as compared to the control sample 54.

The TGA/DTG thermograms represent the thermal stability of the control and treated samples of iron sulphate (Figure4 and Figure5). The TGA thermograms of the control and treated samples exhibited three steps thermal degradation (Figure4 and Table 3). The thermogram pattern was matched with the literature, according to which, the first and second steps of weight loss might occur due to the loss of water molecules from iron sulphate heptahydrate (FeSO₄•7H₂O). Consequently, the final major weight loss takes place due to the oxidation as well as dehydration of the other part of monohydrate, sulphate decomposition 5556. The data represents that the control sample lost weight by 42.8, 6.96, and 23.46% in 1^st, 2^nd and 3^rd steps of degradation, respectively (Table3). However, the Biofield Energy Treated sample lost weight by 40.31, 5.84, and 25.05% in 1^st, 2^nd and 3^rd steps of degradation, respectively (Table3). Thus, it revealed that the weight loss of Biofield Energy Treated sample in the first and second step was reduced by 5.82% and 16.09%, respectively, whereas, in third step, it was increased by 6.78%, compared to the control sample. Also, the overall weight loss of the Biofield Treated sample was reduced by 2.76% as compared to the control sample during this process. It represents that the thermal stability of the Biofield Energy Treated sample was increased as compared to the control iron sulphate sample, which might be due to the increase in particle size of the treated sample 57.

The DTG thermograms of both the control and Biofield Energy Treated samples (Figure 5) exhibited three peaks. The analysis revealed that the maximum thermal degradation temperature (T_max) of 1^st and 4^th peaks of the Biofield Energy Treated sample were reduced by 2.57% and 0.41%, respectively, as compared to the control sample (Table 4). Besides, the T_max of 2^nd and 3^rd peaks of the Biofield Energy Treated sample were slightly increased by 0.29% and 0.35%, compare to the control sample (Table 4). Overall, the TGA/DTG analysis revealed that the thermal stability of the Biofield Energy Treated iron sulpahte was altered as compared to the control sample, which might be due to the alteration in particle size of the treated sample after the Biofield Energy Treatment.

The DSC thermograms of the control and Biofield Energy Treated samples of iron sulphate are presented in Figure 6. Some studies reported the dehydration behavior of a hydrated iron salt by using DSC and TGA techniques 5556. According to this, the DSC thermogram of iron sulphate heptahydrate shows three peaks, when heated at the rate of 10 °C under nitrogen atmosphere. Among these peaks, firstpeak was observed at temperature <100 °C (FeSO₄•7H₂O→FeSO₄•4H₂O), second peak at 85 to 149 °C (FeSO₄•4H₂O→ FeSO₄•H₂O), and third peak at 247 to 342 °C (FeSO₄•H₂O→FeSO₄) due to the dehydration of water molecules. They also concluded that accurate thermal data from the TGA/DSC dehydration experiments depends on various factors like proper selection of the heating rate, particle size, open or closed pan, etc. 55. Table 5.

In this study, the DSC thermogram of the control iron sulphate heptahydrate (Figure 6) showed the presence of the four endothermic peaks. The 1^st sharp endothermic peak was observed at 75.83 °C, which was may be due to the melting point of iron sulphate heptahydrate. However, the temperature of this peak (69.58 °C ) was significantly decreased in Biofield Energy Treated sample by 8.24% with a significant reduction in the latent heat of fusion (∆H) by 92.29% (Table3), compared with the control sample. The 2^nd broad endothermic peak at 95.06 °C in control sample was might be due to the dehydration of two molecules of water from FeSO₄•6H₂O. Further, the temperature of this peak was also significantly reduced by 19.29% in the Biofield energy Treated sample (76.72°C) along with reduction in ∆H by 86.29%, compared with the control sample. Later on, the melting temperature of the 3^rd sharp endothermic peak (due to removal of three water molecules) was increased in the Biofield Energy Treated sample (121.10°C) by 4.57% with a significant 60.92% increase in ∆H, compared with the control sample (115.81 °C). Finally, a broad endothermic peak was observed at 273.13°C in the control sample (due to dehydration of iron sulphate in anhydrous form), which was slightly reduced by 0.61% in the Biofield Energy Treated sample (271.48°C) along with 6.37% enhancement in ∆H as compared with the control sample (Table3). The overall latent heat of fusion on the Biofield Energy Treated sample (1095.34 J/g) was significantly increased by 15.49% compared with the control sample (948.39 J/g). The alteration in the latent heat of fusion may be due to the changes in the molecular chains and crystal structures 5557. Thus, it is assumed that the Biofield Energy Treatment may alter the thermal stability of iron sulphate by impacting the crystal morphology, molecular chain structures and particle sizes.