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RESULTS AND DISCUSSION
Identification 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 189
0
C-193
0
C, 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.





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Table: Solubility of CBZ in various solvents.
Deionized water Practically insoluble
(17.78mg/ml)
Ether Insoluble
Dichloromethane Freely
Methylene chloride Freely
Ethanol Sparingly
Acetone Sparingly
Chloroform Sparingly
Methanol Sparingly

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.
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Table : Details of FTIR spectrum of CBZ.
Sr.No. Frequency (cm
-1
)
Assignment
1 3455.20 NH stretching
2 3161.43 NH stretching
3 1678.13 -C=O stretching
4
1604.83
-C=C- stretching &
C=O vibration
5 1595.18 -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 CBZ
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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 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.









0
0.19
0.39
0.59
0.745
0.957
y = 0.0478x
R = 0.9989
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20 25
A
b
s
o
r
b
a
n
c
e

Concentration
CALIBRATION CURVE OF CBZ
ABS.
Linear (ABS.)
Sr.no
CONC. ABS.
1
0 0
2
4 0.19
3
8 0.39
4
12 0.59
5
16 0.745
6
20 0.957
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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 Crystal
Melting
Point (
0
C)
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.


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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 D
Sr.
No.
Sample Bulk density
(gm/cc)
Tapped Density
(gm/cc)
1 CBZ Anhydrate 0.5700.76 0.7400.24
2 CBZ Dihydrate 0.400.16 0.5300.15

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 ()
1 CBZ Anhydrate 24.250.14
2 CBZ 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 (%):
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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
Carrs
Consolidaton
Index (%)
1 CBZ Anhydrate 22.970.24
2 CBZ Dihydrate 24.530.15


Hausners ratio:

Table : Hausners ratio of CBZ A & CBZ D.
Sr.
No.
Sample Hausners
Ratio
1 CBZ Anhydrate 1.2980.1
2 CBZ Dihydrate 1.3250.78

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
(%)
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1 CBZ Anhydrate 36.11
2 CBZ 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.
Samples Solubility
(mg/L)
1 CBZ Anhydrate 19.2
2 CBZ Dihydrate 17.9

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 dihydrate
crystals:
Morphology (optical and electron microscopy):
Motic Microphotographs of crystals and crystal size:
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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
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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.
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Figure : X-ray diffractogram of CBZ A crystals
Figure : X-ray diffractogram of CBZ D crystals
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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
value
2-Theta (2)
6.90149 12.041
5.22183 16.966
4.95991 17.869
4.71053 18.823
4.0839 21.749
2.22196 40.568
2.07887 43.498
2.03289 44.534
1.96312 46.206
1.94032 46.701
1.83844 49.541
1.70991 53.55
1.60948 57.188
1.51256 61.230


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.

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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.
100.00 200.00
Temp [C]
-40.00
-30.00
-20.00
-10.00
0.00
mW
DSC
188.78x10
0
C Onset
198.24x10
0
C Endset
191.37x10
0
C Peak
-1.50x10
0
J Heat
-42.19x10
0
mW Hei ght
170.80x10
0
C Onset
177.03x10
0
C Endset
174.06x10
0
C Peak
-167.22x10
0
mJ Heat
-7.46x10
0
mW Hei ght
Fi l e Name: Anhydrous CBZ.tad
Detector: DSC-60
Sampl e Name:Anhydrous CBZ
Sampl e Wei ght: 5.000[mg]
Cel l : Al umi num Seal
Atmosphere: Ni trogen
Fl ow Rate: 50[ml /mi n]
Annotati on:
[Temp Program]
Temp RateHol d TempHol d Ti meGas
[C/mi n ] [ C ] [ mi n ]
10.00 250.0 0
Thermal Analysis Result of Anhydrous CBZ
Anhydrous CBZ.tad DSC


Figure: DSC thermograph of CBZ anhydrate
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50.00 100.00 150.00 200.00
Temp [C]
-30.00
-20.00
-10.00
0.00
mW
DSC
74.33x10
0
C Onset
102.56 x10
0
C Endset
90.05x10
0
C Peak
-4.45x10
0
J Heat
-29.80x10
0
mW Height
164.23 x10
0
C Onset
173.80 x10
0
C Endset
169.36 x10
0
C Peak
-89.48x10
0
mJ Heat
-2.25x10
0
mW Height
192.94 x10
0
C Onset
202.68 x10
0
C Endset
195.22 x10
0
C Peak
-1.43x10
0
J Heat
-30.96x10
0
mW Height
File Name: CBZ Hydrous.tad
Detector: DSC-60
Sample Name: CBZ HYDROUS
Sample Weight: 5.000[mg]
Cell: Aluminum Seal
Atmosphere: Nitrogen
Flow Rate: 50[ml/min]

Figure: DSC thermograph of CBZ dihydrate

Table : DSC thermogram data for CBZ A and CBZ D.

Sr.No

Sample
Enthalpy
of Fusion
(J/g)
Onset
Temperature
(
0
C)
Peak
Temperature
(
0
C)
Endset
Temperature
(
0
C)

1. CBZ-
anhydrate


167.2

188.7

191.30

198.20
2. CBZ dihydrate
(1
st
peak)


89.48

164.2

169.3

173.8
3. CBZ dihydrate
(2
nd
peak)


1.4

192.9

195.20

202.6

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 191.30
0
C (Table 7.11).
While the DSC thermograms of CBZ dihydrate shows more than one
endothermic peaks, one at higher melting point i.e. 195.20
0
C and one
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at lower melting point i.e.169.30
0
C, 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 anhydrate
Table: Details of FTIR spectrum of CBZ A.
Sr.
No.
Frequency (cm
-1
)
Assignment
1 3466.14 NH stretching
2 3160.42 NH stretching
3 1677.13 -C=O stretching
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4
1604.83
-C=C- stretching &
C=O vibration
5 1595.18 -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
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1 3465.14 NH stretching
2 3193.21 NH stretching
3 1678.13 -C=O stretching
4
1604.83
-C=C- stretching &
C=O vibration
5 1595.18 -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

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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. Concentration Absorbances
y = 0.0477x
R = 0.9984
0
0.1
0.2
0.3
0.4
0.5
0.6
0 5 10 15
A
b
s
o
r
b
a
n
c
e
s

Concentration
Standard calibration curve of CBZ in distilled
water
Absorbances (nm)
Linear (Absorbances
(nm))
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No.
(g/ml) (nm)

1 0 0.000
2 2 0.098
3 4 0.194
4 6 0.292
5 8 0.389
6 10 0.490


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
1 0
0.000 0.000
2 30
17.21 5.99
3 60
22.99 11.51
4 90
28.47 16.64
5 120
35.22 20.68
6 150
40.97 28.19
7 180
46.42 33.37
8 210
51.7 38.48
9 240
56.87 43.7

The initial powder dissolution profiles of the two crystal forms of
carbamazepine indicate that the anhydrate form had a significantly
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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
1 0 0.00 0.00
2 10
11.538 2.215
3 20
13.532 3.732
4 30
17.024 5.927
5 40
18.815 7.985
6 50
20.565 9.425
7 60
22.479 11.255
8 70
24.168 13.173
9 80
25.726 14.484
0
10
20
30
40
50
60
0 100 200 300 400 500
%

D
R
U
G

R
E
A
L
E
A
S
E

TIME
POWDER DRUG DISSOLUTION
A-CBZ
H-CBZ
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10 90
27.519 16.088
11 100
29.199 17.798
12 110
31.288 19.505
13 120
33.689 19.729
14 130
33.613 21.615
15 140
35.154 23.601
16 150
38.642 26.769
17 160
39.993 28.139
18 170
41.636 29.699
19 180
43.24 31.083
20 190
44.564 32.43
21 200
46.14 34.098
22 210
47.541 35.389
23 220
48.586 36.861
24 230
50.114 38.246
25 240
51.619 39.661


Figure 7.19: Powder Dissolution of CBZ A & CBZ D for 4 Hrs.
0
10
20
30
40
50
60
0 50 100 150 200 250 300
%

D
R
U
G

R
E
A
L
E
A
S
E

TIME
A-CBZ
H-CBZ
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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.