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RESULTS AND DISCUSSIONIdentification test for Carbamazepine:

Color test: Carbamazepine (CBZ) was identified using a colorimetric test in which ammonium molybdate was the reagent of choice yields a faint blue color. An additional colorimetric test with 2mL nitric acid in a water bath for three minutes yields an orange color. Which complies with color reported in United States Pharmacopoiea.

Crystal test: CBZ was identified using a crystallization test, where needle-shaped crystals wereformed when CBZ was dissolved in a lead iodide solution, complies with U.S.P specification.

Melting point: The melting point of the CBZwas found to be 1890C-1930C, which complies with melting point reported in United States Pharmacopoiea.

Solubility study:The solubility of CBZ was assessed in different solvents and obtained data shown in (Table 7.1). Disappearance of the solid particle was considered as solubility.

Table: Solubility of CBZ in various solvents.Deionized waterPractically insoluble (17.78mg/ml)

EtherInsoluble

DichloromethaneFreely

Methylene chlorideFreely

EthanolSparingly

AcetoneSparingly

ChloroformSparingly

MethanolSparingly

The drug sample of CBZ was found to be insoluble in water and freely and sparingly in the above mentioned solvents which complies with the solubility reported in United States Pharmacopoiea.

Fourier transform infrared absorption spectrophotometry: All the prominent and primary peaks were observed in FTIR spectrum of CBZ(Fig:7.1) and matched with the reference spectrum as per United States Pharmacopoiea.

Figure : FTIR Spectrum of CBZ.Table : Details of FTIR spectrum of CBZ.Sr.No.Frequency (cm-1)Assignment

13455.20NH stretching

23161.43NH stretching

31678.13-C=O stretching

41604.83-C=C- stretching &C=O vibration

51595.18-NH deformation

The above frequencies of the peaks were found to be concordant with the FT-IR spectra of pure CBZ mentioned in United States Pharmacopoiea, confirms qualitative standard of sample used for the purpose.

Determination of max for CBZ by UV spectroscopy: The max of CBZ (Fig:7.2)was found to be 285nm, which complies with the specification of USP.

Figure : UV Spectrum of CBZmax of any substance in respective are peculiar properties of that substance hence are useful for qualitative & quantitative standardization of substance.

Standard calibration curve of CBZ in ethanol: As described earlier, standard calibration curve was prepared for concentration of 1g/ml to 5g/ml at 285 nm. The graph of absorbance v/s concentration was plotted and data were subjected to linear regression analysis. The standard calibration curve of drug in ethanol was as depicted in (Fig:7.3). The data of absorbance is as shown in table. The data had correlation coefficient of 0.997 and equation of regressed line is y = 0.054x + 0.006.

Table : Standard calibration curve of CBZ in ethanol.

Sr.noCONC.ABS.

100

240.19

380.39

4120.59

5160.745

6200.957

Figure : Standard calibration curve of CBZ in ethanol

Evaluation of CBZ anhydrate& CBZ dihydrate crystals. Physical property:

Melting point: Melting occurs when a temperature is reached at which the thermal energy of the particles is great enough to overcome the intracrystalline forces that hold them in position. Melting point of CBZ anhydrate&dihydrate crystals are depicted in (Table 7.4). Table : Melting point of the CBZ A& CBZ D crystals Sr.no. Types of CrystalMelting Point (0C)

1 CBZ Anhydrate 184-190

2 CBZ Dihydrate 186-192

From the data obtained for melting point determination, it reveals that changes in both crystal forms CBZ anhydrate and CBZ dihydrate, did not show significant change in melting point. Loss on drying: Complete hydration of the anhydrate samples was confirmed by the loss on drying method. The water content of the anhydrate CBZ was 0.05% w/w of where as the, dihydrate samples had a loss on drying of 12.1% w/w, These values are near to the stoichiometric value calculated for the water content of CBZ dihydrate (13.2% wt/wt) reported in literature. The weight loss is attributed to the dehydration of CBZ dihydrate, i.e. removal of lattice water.

Micromeritic properties:

Bulk density and Tapped density: Bulk density and Tapped density are the relative measures of interparticulate interactions. In a free flowing powder such interactions are generally less and the value of both densities will remain close to each other.Table : Bulk density and Tapped density of CBZ A& CBZ DSr. No.Sample Bulk density (gm/cc)Tapped Density (gm/cc)

1CBZ Anhydrate0.5700.760.7400.24

2CBZ Dihydrate0.400.160.5300.15

From the data (Table 7.5), it was found that CBZ anhydrate& CBZ dihydrate possessed good flow property.

Angle of repose () / Flowability:

Table : Angle of repose of CBZ A & CBZ D.Sr.No.Sample Angle of Repose ()

1CBZ Anhydrate 24.250.14

2CBZ Dihydrate 33.930.90

From the data (Table 7.6), angle of repose was found to be < 40 i.e. CBZ anhydrate& CBZ dihydrate possessed good flow property.

Measure of powder compressibility: Carrs consolidation index (%): CBZ anhydrate crystals were proven to be excellent compressible according to Carrs consolidation index (Table 7.7) as compared to CBZ dihydrate.

Table: Carrs Consolidation Index of CBZ A & CBZ D.Sr. No.Sample CarrsConsolidatonIndex (%)

1CBZ Anhydrate22.970.24

2CBZ Dihydrate24.530.15

Hausners ratio:

Table : Hausners ratio of CBZ A & CBZ D.Sr. No.Sample Hausners Ratio

1CBZ Anhydrate 1.2980.1

2CBZ Dihydrate1.3250.78

CBZ anhydrate crystals were proven to be acceptably compressible according to Hausners ratio (Table 7.8) as that of CBZ dihydrate had Hausners ratio > 1.21.

Porosity (%): Table : Porosity of CBZ A & CBZ D .Sr. No.Sample Porosity (%)

1CBZ Anhydrate 36.11

2CBZ Dihydrate 16.66

From the data (Table 7.9), it was observed CBZ dihydrate had closed packing arrangement with different particle size distribution, which indicates the poor dissolution property. From the all above micromeretics data, it was observed that CBZ dihydrate had poor flow property, poor compressibility and closed packing arrangement which indicate poor dissolution property in comparison to that of CBZ anhydrate .

Solubility study: The solubility of both the CBZ crystals of anhydrate&dihydrate was measured in water by UV spectrophotometry at 285 nm. CBZ anhydrate and dihydrate solubility was found to be 19.2 mg/L and 17.9 mg/L respectively. Table: Solubility of the CBZ A & CBZ D.Sr. No.SamplesSolubility (mg/L)

1 CBZ Anhydrate19.2

2 CBZ Dihydrate17.9

The set of observations mentioned in (Table 7.10)were proven thatthe CBZ anhydrate crystals showed increased solubility as compared to the CBZ dihydratecrystals, might be due to intermolecular hydrogen boning associated with crystal water.Characterization of CBZ anhydrate&CBZ dihydratecrystals:Morphology (optical and electron microscopy): Motic Microphotographs of crystals and crystal size: The photographs of CBZ anhydrate& CBZ dihydrate crystals captured by using motic microscope shown in (Fig: 7.4 & Fig: 7.5). The significant differences in the external morphology of 3D crystals cannot be predicted exactly with the 2D system. Photographs give an idea about the influence of all variables on the external appearance of the crystals i.e. crystal habit.

Figure 7.4: Motic Microphotographs of CBZ anhydrate

Figure : Motic Microphotographs of CBZ dihydrate From the above Motic Microphotographs (Fig: 7.4 & Fig: 7.5), CBZ anhydrate shown more prominent crystal habit, the crystals appeared needle shaped. While CBZ dihydrate crystals appeared to be short and thin needle, might be due to the occurrence of whiskering during the formation of dihydrate crystals.

X-Ray Powder Diffraction study (XRPD): The diffraction pattern generated when X-rays pass through the 3-D arrangement of atoms in a crystalline structure is used to identify and characterize unknown crystalline material. By observing position of peaks i.e. 2-Theta (2), intensity of peaks (Height), appearance or disappearance of peaks and d-spacing, confirmation of crystals are predicted. Figure : X-ray diffractogram of CBZ A crystalsFigure : X-ray diffractogram of CBZ D crystals The experimentally obtained XRD patterns for the characterisation of crystals of carbamazepine i.e. anhydrate CBZ crystals and dihydrate CBZ crystals presented in (Fig: 7.8 & 7.9). XRD pattern shown that both the anhydrate and dihydrate CBZ crystals had same crystal structure i.e. polymorphic form III (P-monoclinic), as per U.S.P specified values of 2-Theta. The XRD patterns obtained for both the crystals CBZ A& CBZ D and from the value of d-spacing and 2, peaks disappearance observed in XRD pattern of CBZ dihydrate as compared with the XRD pattern of CBZ A. The peak disappeared of d-spacing and 2 value as follows:d-spacing value2-Theta (2)

6.9014912.041

5.2218316.966

4.9599117.869

4.7105318.823

4.083921.749

2.2219640.568

2.0788743.498

2.0328944.534

1.9631246.206

1.9403246.701

1.8384449.541

1.7099153.55

1.6094857.188

1.5125661.230

The disappearance of peaks in CBZ D indicated that, the peak disappearance observed might be due to inclusion of water molecule in the crystal lattice of CBZ dihydrate crystals.

7.3.4 Differential Scanning Calorimetry (DSC): Differential scanning calorimetry (DSC) is a thermo analytical technique in which the difference in the amount of heat required to increase the temperature of a sample and reference are measured as a function of temperature. DSC was performed in order to assess the thermotropic properties and thermal behaviour of the drug (CBZ) and its crystals prepared.

Figure: DSC thermograph of CBZ anhydrate

Figure: DSC thermograph of CBZ dihydrate

Table : DSC thermogram data for CBZ A and CBZ D.Sr.No SampleEnthalpy of Fusion (J/g)OnsetTemperature (0C)PeakTemperature (0C)EndsetTemperature (0C)

1. CBZ- anhydrate

167.2188.7191.30198.20

2.CBZ dihydrate(1st peak)

89.48164.2169.3173.8

3.CBZ dihydrate(2nd peak)

1.4192.9195.20202.6

DSC thermograms of CBZ anhydrate& CBZ dihydrate crystals were shown in (Fig: 7.10 & 7.11). The DSC thermogram of CBZ anhydrate crystals shows only one endothermic peak at 191.300C (Table 7.11). While the DSC thermograms of CBZ dihydrate shows more than one endothermic peaks, one at higher melting point i.e. 195.200C and one at lower melting point i.e.169.300C, which might be due to association of crystal water i.e. intermolecular hydrogen bonding with N-H.

Fourier transform infrared spectrophotometry (FTIR): FTIR spectrum of CBZ anhydrate: FT-IR spectra of CBZ anhydrate observed the prominent peak at 1675 cm-1 might be due to the carbonyl nC-0, and the band at 3455.5 cm-1 due to the intermolecular hydrogen-bonded nNH.Figure: FTIR Spectrum of CBZ anhydrateTable: Details of FTIR spectrum of CBZ A.Sr. No.Frequency (cm-1)Assignment

1 3466.14NH stretching

23160.42NH stretching

31677.13-C=O stretching

41604.83-C=C- stretching &C=O vibration

51595.18-NH deformation

FTIR spectrum of CBZ dihydrate: FT-IR spectra of CBZ anhydrate observed the prominent peak at 1678.13 cm-1 might be due to the carbonyl nC-0, and the band at 3439.5cm-1 due to the intermolecular hydrogen-bonded nNH.Figure 7.13: FTIR Spectrum of CBZ dihydrate

Table : Details of FTIR spectrum of CBZ D.Sr. No.Frequency (cm-1)Assignment

13465.14NH stretching

23193.21NH stretching

31678.13-C=O stretching

41604.83-C=C- stretching &C=O vibration

51595.18-NH deformation

The FTIR spectrum of CBZ anhydrate and dihydrate crystals shown in (Fig: 7.12, 7.13) respectively. It was observed that the nNH stretching band at 3455.5 cm-1 of CBZ anhydrate crystals shifted to 3439.5 cm-1 in CBZ dihydrate crystals (Table:7.12), might be due to intermolecular hydrogen bonding with N-H in case of CBZ dihydrate.

Determination of max for CBZ by UV spectroscopy: The max of CBZ in dissolution media i.e. Distilled Water + 1% SLS was found to be 285nm, which complies with the specification of USP (Figure 7.16).

Figure : UV Spectrum of CBZ in Distilled Water + 1% SLS

max of any substance in respective are peculiar properties of that substance hence are useful for qualitative & quantitative standardization of substance.

Standard calibration curve of CBZ:Standard calibration curve of CBZ in solution of distilled water + 1% SLS:Standard calibration curve was prepared for concentration of 2g/mL to 10g/mL at 285 nm. The graph of absorbance v/s concentration was plotted and data were subjected to linear regression analysis. The standard calibration curve of drug in ethanol was as depicted in (Figure 7.17). The data of absorbance is as shown in table. The data had correlation coefficient of 0.9984 and equation of regressed line is y = 0.0477X

Table : Standard calibration curve of CBZ in distilled water + 1% SLS.Sr. No.Concentration(g/ml)Absorbances(nm)

100.000

220.098

340.194

46 0.292

580.389

6100.490

Figure : Standard calibration curve of CBZ in distilled water + 1% SLS.

Powder dissolution studies:Table:Powder dissolution study carried out initially for 3 min. Sr. No.Time in seconds% Release of CBZ A% Release of CBZ D

100.0000.000

23017.215.99

36022.9911.51

49028.4716.64

512035.2220.68

615040.9728.19

718046.4233.37

821051.738.48

924056.8743.7

The initial powder dissolution profiles of the two crystal forms of carbamazepine indicate that the anhydrate form had a significantly higher dissolution rate than the dihydrate, essentially reaching maximum concentration within 30 seconds (Fig.7.18).

Figure 7.18: Powder Dissolution of CBZ A & CBZ D

Table: Powder dissolution study continued for 4 hours.Sr. No.Time in seconds% Release of anhydrate CBZ% Release of Dihydrate CBZ

100.000.00

21011.5382.215

32013.5323.732

43017.0245.927

54018.8157.985

65020.5659.425

7 6022.47911.255

8 7024.16813.173

9 8025.72614.484

10 9027.51916.088

11 10029.19917.798

12 11031.28819.505

13 12033.68919.729

14 13033.61321.615

15 14035.15423.601

16 15038.64226.769

17 16039.99328.139

18 17041.63629.699

19 18043.2431.083

20 19044.56432.43

21 20046.1434.098

22 21047.54135.389

23 22048.58636.861

24 23050.11438.246

25 24051.61939.661

Figure 7.19: Powder Dissolution of CBZ A & CBZ D for 4 Hrs.

The powder dissolution profiles of the two crystal forms of carbamazepine indicate that the anhydrate form had a significantly higher dissolution rate than the dihydrate, essentially reaching maximum concentration within 140 min (Fig 7.19). However, it was found that almost immediately after a peak concentration was reached, the percent dissolution started to decline, This observation were attributed to the crystallization of hydrate that has a significantly lower solubility. Interestingly, this solution-mediated phase transitionwas visually apparent during the dissolution test. Following addition of anhydrate powder to the dissolution vessel, the solid particles dissolved rapidly, resulting in a nearly clear solution that subsequently turned turbid within minutes. The results indicate that the transformation from anhydrate CBZ to dihydrate CBZ to the more stable hydrate is very rapid in aqueous medium.

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