Growth and Characterization of 4-carboxyanilinium Dihydrogen Phosphate Semi-organic Complex Crystal

MATERIALS AND METHODS

The 4-aminobenzoic acid, orthophosphoric acid, ethanol and deionized water were used in the crystal growth process of title compound and they were purchased from Sigma Aldrich Company, India. The single crystal of 4-carboxyanilinium dihydrogen phosphate was obtained by slow evaporation technique at room temperature from the aqueous ethanol solution of 4-aminobenzoic acid mixed with aqueous solution of orthophosphoric acid in 1:1 stoichiometric ratio.

Fig. 1:	Grown crystal of 4-carboxyanilinium dihydrogen phosphate

This solution was stirred well for 1 h and after filtering process it was poured into the petri disc. After a period of 15 days, rod like crystals of title compound was harvested and which is shown in Fig. 1. The single crystal Bruker SMART APEX CCD X-ray diffractometer with Mo Kα radiation (λ = 0.71073 Å) was used to carry out the unit cell dimension and space group. This data was taken from SAIF, IIT Madras, Chennai, Tamil Nadu, India. The XPERT–PRO X–ray diffractometer with Cu Kα (λ = 1.54060 Å) radiation was used to record the powder diffraction pattern in CSIR-CECRI, Karaikudi, Tamil Nadu, India. The FTIR vibrational spectrum was recorded using SHIMADZU FTIR spectrometer in the range 4000-400 cm^–1. Also, the FT-Raman spectrum was recorded in SAIF, IIT Madras, Chennai using the BRUKER: RFS 27 Raman spectrometer in the wavenumber range 4000-400 cm^–1. The optical absorption spectrum of 4-carboxyanilinium dihydrogen phosphate crystal has been recorded with SHIMADZU-UV1800 double beam spectrometer in the wavelength range 200-1100 nm in steps of 1 nm at V.H.N.S.N. College, Virudhunagar, Tamil Nadu, India.

The surface morphology and elemental analysis has been carried out by CARLZEISS EVO18 scanning electron microscope at CSIR-CECRI, Karaikudi, Tamil Nadu, India. Using the disc diffusion method, the antimicrobial activity of parent and its complex crystals were tested against 4 different kinds of microorganisms in Sri Kaliswari College, Sivakasi, Tamil Nadu, India.

RESULTS AND DISCUSSION

Single crystal XRD: The unit cell parameter and space group of 4-carboxyanilinium dihydrogen phosphate crystal was obtained using the SMART APEX CCD area detector diffractometer. Initially, these are checked with the Cambridge Structural Database (CSD) for confirmation. The report shows that the title crystal was exactly matched with already reported values⁷. The crystallographic data of 4-carboxyanilinium dihydrogen phosphate crystal is shown in Table 1 and was compared with already reported values⁷. The molecular structure of title crystal is illustrated in Fig. 2.

Fig. 2:	Molecular structure of 4-carboxyanilinium dihydrogen phosphate

The single crystal XRD study revealed that the grown crystal was a complex one, since the one proton of phosphoric acid was liberated and joined with 4-carboxyaniline NH₂ group, then it becomes as 4-carboxyaniline cation and dihydrogen phosphate anion. Also this study confirmed the grown crystal crystallizes in the monoclinic system with space group P2₁/n.

Powder XRD analysis: The powder X-ray diffraction patterns of 4-carboxyanilinium dihydrogen phosphate crystal is shown in Fig. 3 which was recorded using XPERT-PRO X-ray diffractometer with Cu Kα (λ = 1.54060 Å) radiation from 10 80̊ at 10̊/sec scan step time. The diffracted peaks are indexed using INDX software. The good crystalline nature and purity of the grown crystal was observed from the sharp intensity PXRD pattern.

Table 1:	Crystallographic data of 4-carboxyanilinium dihydrogen phosphate crystal

Fig. 3:	Diffraction patterns for 4-carboxyanilinium dihydrogen phosphate crystal

Fig. 4:	FTIR spectrum for 4-carboxyanilinium dihydrogen phosphate crystal

The Debye-Scherrer equation was used to determine the average crystalline size of 4-carboxyanilinium dihydrogen phosphate crystal, which can be written as:

The dislocation density can be calculated from, following formula:

For the 4-carboxyanilinium dihydrogen phosphate crystal, the average crystalline size is found to be as 54 nm. The dislocation density of this crystal is determined as 3.43×10¹⁴ m².

Vibrational analysis: The FTIR and FT-Raman spectra of 4-carboxyanilinium dihydrogen phosphate complex crystal are shown in Fig. 4 and 5, respectively and their corresponding IR and Raman assignments are listed in Table 2. The title compound has –[NH₃]⁺, -COOH, para substituted benzene ring and H₂PO₄^‾ functional groups.

-[NH₃]⁺cation vibration: Normally, -[NH₃]⁺ group has C₃v symmetry in the free state with a pyramidal structure. The normal modes of vibrations of this group occurs as ν₁ (A₁), ν₂ (A₁), ν₃ (E) and ν₄ (E)¹². Among these vibrations, the symmetry stretching and bending modes (ν1 and ν2) are non-degenerative whereas the antisymmetric stretching and bending modes (ν₃ and ν₄) are doubly degenerated. All these modes are both IR and Raman active¹². The antisymmetric and symmetric stretching modes of -[NH₃]⁺ group are expected in the region of 3200 and 2800 cm^–1, respectively^12,13. Also, the antisymmetric and symmetric deformation modes are expected to appear in the region of 1660-1610 and 1550-1480 cm^–1, respectively¹³. The -[NH₃]⁺antisymmetric stretching wavenumber of title crystal was observed as strong band at 3233 cm^–1 in IR and as a weak band at 3226 cm^–1 in Raman spectra and the stretching wavenumber of this group was identified at 2810 cm^–1 (IR), 2831 cm^–1 (Raman), respectively.

The antisymmetric bending mode was identified at 1655 cm^–1, 1612 cm^–1 in IR and at 1649 cm^-1, 1616 cm^–1 in Raman spectra for the title compound, respectively. Also, symmetric bending mode was attributed at 1580 cm^–1, 1541 cm^–1 in IR and at 1525 cm^–1 in Raman spectra, respectively.

Fig. 5:	FT-Raman spectrum for 4-carboxyanilinium dihydrogen phosphate crystal

Table 2:	Wavenumber assignments for 4-carboxyanilinium dihydrogen phosphate crystal in FTIR and FT-Raman spectra
w: Weak, s: Strong, br: Broad, m: Medium, υ: Stretching, υs: Symmetric stretching, υas: Anti symmetric stretching, δs: Symmetric bending, δas: Antisymmetric bending, γ: Out-of-plane bending, ß: In-plane bending, ρ: Rocking, t: Torsional, ω: Wagging

In the present study, the strong bands observed at 1138 cm^–1 in IR and at 1134 cm^–1 in Raman spectra was assigned to the rocking mode of –[NH₃]⁺group. The –[NH₃]⁺ wagging mode was observed as a strong band at 961 cm^–1 in the FTIR spectrum only. The torsional mode of –[NH₃]⁺ group was also identified in the range of 760-750 cm^{–1 14,15}. In present case, the torsional mode vibration was observed at 758 cm^–1 as a medium band in the IR spectrum only. The broad band centered at 3000 cm^–1 in IR spectrum was attributed to the presence of extensive 3 dimensional hydrogen bonding network that exists in the crystal.

Carboxylic group vibration: The antisymmetric and symmetric stretching vibrational mode of C=O group in carboxylic acid have wavenumbers in the region 1720-1680 cm^–1 and 1680-1640 cm^–1, respectively¹³. In the present study, ν_as (C=O) mode occurs at 1655 cm^–1 in IR and at 1649 cm^–1 Raman spectra. Also, bands observed at 1612 cm^–1, 1616 cm^–1 in both spectra was assigned to the ν_s (C=O) mode for the title crystal. The ν (C-O) mode of carboxylic group normally occurs in the vibrational region of 1320-1210 cm^–116. The title compound has wavenumbers at 1325, 1215 cm^–1 in IR and at 1324 cm^–1, 1214 cm^–1 in Raman spectra was attributed to ν (C-O) mode. The O-H stretch from CO-OH group was identified at 3065-2826 cm^{–1 15,17}. It was attributed at 3071, 2810 cm^–1 in IR and at 3095, 3065, 3018, 2831 cm^–1 in Raman spectra, respectively for title compound.

Table 3:	Vibrational distribution of [PO4]3- ion to [H2PO4]‾ ion

The in-plane and out-of plane bending wave numbers of O-H group appears in the region between 1440-1395 cm^–1 and 960-875 cm^–1, respectively^15,17. In the present study, β(O–H) mode was identified as medium intensity bands at 1454 cm^–1, 1425 cm^–1 in IR and at 1445 cm^–1 in Raman spectra. Also γ (O-H) mode was attributed only at 961 cm^–1, 891 cm^–1 in IR spectrum and at 893 cm^–1 in Raman spectrum, respectively for title compound.

Para substituted benzene ring vibration: The C-H stretching mode for para substituted benzene ring was expected in the region 3115-3005 cm^{-1 12,15}. In this case, the bands observed at 3071cm^-1 in IR and at 3095, 3065, 3018 cm^–1 in Raman spectra was assigned to ν (C-H) mode. The C-H in-plane and out-of-plane bending vibrations are seen in the range 1250-1000 and 900-690 cm^–1, respectively¹⁵. For the title compound, the β(C-H) mode was identified at 1215, 1138, 1067 cm^–1 in FTIR and at 1214, 1182, 1134, 1007 cm^–1 in FT-Raman spectra, respectively. The γ (C-H) mode was identified at 891, 758, 687 cm^–1 in IR spectrum and at 893 cm^–1 in Raman spectrum for the title compound. The C = C and C-C ring stretching modes occur in the region 1650-1430 and 1400-1300 cm^–1, respectively¹³. For the title compound ν (C = C) mode was assigned at 1612, 1580, 1541, 1512 cm^–1 in IR and 1616, 1525 cm^–1 in Raman spectra. Also ν (C-C) mode was observed at 1325 cm^–1, 1302 cm^–1 in IR spectrum. The C-N stretching mode was identified at 1325 cm^–1 in spectrum for this title compound. The ring breathing mode was identified at 853 cm^–1 in IR and 836 cm^–1 in Raman spectra for the title compound.

Dihydrogen phosphate anion vibration: The phosphate ion has T_d symmetry in the free state which has symmetric stretching ν₁(A₁), antisymmetric stretching ν₃(F₂), symmetric bending ν₂(E) and antisymmetric bending ν₄(F₂) internal vibrational modes. These wavenumbers occur at 936 cm^–1 (ν_sym), 420 cm^–1 (δ_sym), 1004 cm^–1 (ν_asym) and 573 cm^–1 (δ_asym)^18,19. Due to the presence of two P-OH bonds, the molecular symmetry of [H₂PO₄]^-ion reduces to C_2v symmetry. As a result, the degeneracy of the modes ν₂, ν₃ and ν₄ was fully lifted: ν₂ splits into two components (A1+A2) and ν₃ and ν₄ split into three components (A₁+B₁+B₂) each¹⁸. The symmetric stretching mode (A₁) normally occurs at 880 cm^–1 and the symmetric bending mode E splits into two, appearing at 385 (A₁) and 373 (A₂) cm^–1. The antisymmetric stretching mode (F₂) splits into three occurring at 1090 (A₁), 1123 (B₁) and 940 (B₂) cm^–1. The antisymmetric bending mode (F₂) has also lost its degeneracy and was expected to occur at 525 (A₁), 510 (B₁) and 503 (B₂) cm^–1. All the species are both infrared and Raman active except for A₂ species, which was only Raman active¹⁹.

In the present study, the antisymmetric stretching mode of [H₂PO₄]^‾ ion was identified at 1067(A₁), 1138(B₁), 961(B₂) cm^–1 and the symmetric stretching mode was observed in IR and Raman spectra at 891(A₁), 893(A₁) cm^–1, respectively. Also, the symmetric bending mode of this group was attributed at 387, 374 cm^–1 in Raman spectrum only and antisymmetric bending mode was identified at 544(A₁), 434(B₁) and 488(B₂) cm^–1 in IR spectrum only. All these assignments confirm the existence of dihydrogen phosphate anion in the C_2v symmetry and it is clearly depicted in Table 3. The P-OH group has a strong band in the region 1040-810 cm^–1 which was due to the stretching of P-OH bands²⁰. The medium bands occur at 891, 853 cm^–1 in IR sand at 893 cm^–1 in Raman spectra was assigned to P-OH stretching vibration of title crystal. The in-plane and out-of-plane bending vibrations occur in the region 1350-1200 and 950-750 cm^–1, respectively^19,20. For the title compound these modes are identified in IR spectrum at 1215, 891 cm^–1 and in Raman spectrum at 1214, 893 cm^–1, respectively. The stretching vibrations of P=O generally give rise to strong band in the region 1300-1140 cm^–1 for dihydrogen phosphate. The absorption bands at 1185, 1182 cm^–1 in both spectra is assigned to P=O stretching mode of 4-carboxyanilinium dihydrogen phosphate crystal.

Optical analysis: 4-carboxyanilinium dihydrogen phosphate single crystal absorbance spectrum is obtained using SHIMADZU-UV1800 double beam spectrometer in the wavelength range 200-1100 nm. The absorbance spectrum of title crystal is shown in Fig. 6. The lower cut-off wavelength was found to be at 248 nm and at 337 nm. The title crystal has 100% transmittance in the entire visible region which makes usefulness of this material in optical application. The energy gap E_g is determined by using the Tauc’s relation (αhν)² = A(hν- E_g) by plotting the (αhν)²Vs photon energy and extrapolate the linear portion of (αhν)² to the photon energy axis and obtained value is 3.8 eV from the Fig. 7.

SEM with EDX analysis: The morphology and elemental analysis of title compound were performed by SEM with EDX analysis. The microphotograph of title crystal with 4 different magnifications is depicted in Fig. 8. This image reveals that the grown crystal has a smooth surface and flower like morphology. The EDX spectrum for 4-carboxyanilinium dihydrogen phosphate crystal is shown in Fig. 9. The elemental composition of title crystal is shown in Table 4. The EDX analysis reveals that C, N, O and P elements are present in the 4-carboxyanilinium dihydrogen phosphate crystal.

Fig. 6:	Absorbance spectrum for 4-carboxyanilinium dihydrogen phosphate crystal

Antimicrobial activity study: Staphylococcus epidermidis (S. epidermidis), Serratia marcescens (S. marcescens), Methicillin-resistant Staphylococcus aureus (MRSA) and Polyphosphate-accumulating organisms (PAO-1) was used to analyze the antimicrobial activity of 4-carboxyaniline parent and 4-carboxyanilinium dihydrogen phosphate complex crystals by disc diffusion method. The photographic view of bacterial screening for parent and its complex crystals are shown in Fig. 10a and b, respectively. The above mentioned bacterial species were prepared at 50 μg mL^–1 and 100 μg L^–1 concentrations. The measured diameter zone of inhibition of these micro-organisms is shown in Table 5. Both parent and complex crystals show an effective zone of microorganism inhibition if the concentration was increased.

Fig. 7:	Optical band gap for 4-carboxyanilinium dihydrogen phosphate crystal

Fig. 8(a-d):	SEM photograph for 4-carboxyanilinium dihydrogen phosphate crystal, (a) 6000x, (b) 12,000x, (c) 20,000x and (d) 24,000x

Fig. 9:	EDX spectrum for4-carboxyanilinium dihydrogen phosphate crystal

Moreover, the zone of inhibition for the complex crystal was increased due to the presence of phosphoric acid. Especially, it was very dominant against the S. marcescens bacteria. From the Table 5, it is clear that 4-carboxyanilinium dihydrogen phosphate complex crystal was very active against all the organisms except MRSA and PAO-I.

Fig. 10(a-b):	Photographic view showing inhibition region of 4 different micro-organisms (a) 50 μg mL–1 and 100 μg L–1 concentrations against the 4-carboxyaniline parent crystal and (b) 50 μg mL–1 and 100 μg L–1 concentrations against the 4-carboxyanilinium dihydrogen phosphate complex crystal

Table 4:	Elemental composition for 4-carboxyanilinium dihydrogen phosphate crystal

This chemically altered complex molecule may be used to reduce toxicity and increase solubility of drug. It was also given to the human beings in their natural form with the desirable characteristic.

Table 5:	Effective values of inhibited zone for 4-carboxyaniline and 4-carboxyanilinium dihydrogen phosphate crystals
S. epi: Staphylococcus epidermidis, S. mar: Serratia marcescens, MRSA: Methicillin-resistant Staphylococcus aureus, PAO: 1Polyphosphate-accumulating organisms

CONCLUSION

The 4-carboxyanilinium dihydrogen phosphate complex crystal was grown by the slow evaporation technique at room temperature in this study. It has monoclinic crystal system with the space group P2₁/n as suggested by single-crystal X-ray diffraction technique. The average crystalline size of grown crystal is found to be as 54 nm and its PXRD peaks are indexed using the INDX software. Further, the presence of functional groups and elements were verified using FTIR and FT-Raman spectroscopy and energy dispersive X-ray studies. The SEM analyzes shows that the complex crystal has smooth surface and flower like morphology. The optical property of this crystal was analyzed by UV-Visible spectroscopy technique. Also, the optical band gap of title crystal is determined as 3.8 eV. The grown crystals were involved in an antibacterial activity against certain potentially threatening microbes such as Staphylococcus epidermidis, Serratia marcescens, Methicillin resistant Staphylococcus aureus and Polyphosphate accumulating organisms by disc diffusion method which shows that the complex crystal screened the bacteria than that of the parent crystal especially for Staphylococcus epidermidis, Serratia marcescens microorganisms.

SIGNIFICANCE STATEMENT

This study discovers the drug complex crystal that can be beneficial for people in the medicinal field. This study help the researchers to uncover the critical areas of crystal growth in organic-inorganic hybrid that many researchers were not able to explore. Thus a new theory on this complex crystal may be arrived at.

ACKNOWLEDGMENT

The authors sincerely acknowledge their thanks to the Management and Principal of Devanga Arts College, Aruppukottai for their permission and encouragement during their research work.