1.

Synthesis of Phenytoin from Benzil and Urea

Requirements

Chemicals-:

 Benzil
 Urea
 Sodium hydroxide
 Ethanol
 Concentrated hydrochloric acid
 Distilled water

Apparatus:

 Round-bottom flask (100 ml*1)

 Reflux condenser
 Crystallizing dish (500 ml*1)
 Heating mantle
 Stirrer

1.1 Principle:

Base catalyzed reaction between benzyl and urea is used for synthesis of phenytoin.
The reaction Is proceeding via intramolecular cyclization to form an intermediate
heterocyclic pinacol, which on acidification yield hydantoin (phenytoin) as a result
of 1,2-diphenyl shift in pinacol Rearrangement reaction.

1.2 Reaction:

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1.3 Mechanism:

1.4 Pharmacological Use:

1. It is a common antiepileptic drug.

2. Prevention of tonic- clonic seizures.
3. Acute treatment of generalized status epilepticus.]

1.5 Procedure:

1. 5.3 g (0.025 mol) of benzil, 3.0g (0.05 mol) of urea, 15 ml of aqueous sodium
hydroxide solution (30%) and 75 ml of ethanol was placed in a round
bottomed flask of 100 ml capacity.
2. A reflux condenser with the flask was set up and boiled using an electric
heating mantle for 2 h.
3. It was cooled to room temperature, poured the reaction mixture into 125 ml
of water and mixed carefully.
4. The reaction mixture was allowed to stand for 15 min and then it was filtered,
the product under suction to remove an insoluble by-product.
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 5. The strongly acidic filtrate with concentrated hydrochloric acid, was
rendered, then it was cooled in ice-water and immediately filtered off the
precipitated product under suction.
6. It was recrystallized from industrial spirit to obtain about 2.8 g (44%) of pure
5,5-Diphenylhydantoin, m.p. 297-298 °C.

1.6 Calculation:

Here limiting reagent is benzil; hence yield should be calculated from its amount
taken.

1. Molecular formula of benzil = C14H10O2

2. Molecular formula of phenytoin = C15H12N2O2
3. Molecular weight of benzil = 210 g/mole
4. Molecular weight of phenytoin = 252 g/mole

Theoretical yield:

 210 g benzil forms 252 g phenytoin

 Therefore, 5.3 g benzil will form …….? (X) g phenytoin
 X = (252 ×5.3)/210 = 6.36 g
 Theoretical yield = 6.36 g

Practical yield = 1.3g

% Yield = (Practical Yield)/ (Theoretical Yield) × 100

=1.3/6.36 * 100

= 20.6349206

1.7 Conclusion:

Phenytoin was synthesized and the percentage yield was found to be 20.63%.
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 2. Chromatography

2.1 Introduction:

Chromatography is derived from the Greek words “chroma” meaning “colour” and
“graphien” meaningto write. The technique was originally developed by the Russian
Botanist M. S. Tswett in 1903. It is an analytical technique utilized for the
separation, purification and identification of constituents from the mixture. It works
on the principle of differential interaction of solutes with two different phases, viz.,
the stationary phase and the mobile phase. Many modifications were made to the
techniques of chromatography to overcome the shortcomings like analysis time and
the range of compounds that could be detected. Application of pressure was practised
by use of pumps to reduce the time of run. Technologies like spectroscopy and
electrochemical methods were added to enhance detection. With these developments
and modifications, the functional efficiency of chromatographic techniques improved
to a great extent and also the range and type of substances that could be analysed.
2.2 Types of chromatography:
Chromatography is divided into three broad types such as gas, liquid, and
supercritical fluid chromatography. Figure 1 shows the basic classification of
chromatography.

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 TLC

Figure 1: Types of chromatography

Thin layer chromatography

Schraiber, in 1939, developed and employed thin layer chromatography for the first
time. Modern TLC mainly exists as a complementary technique to other column
based liquid chromatographic methods to provide additional information in
separations (multi-modal separation techniques). TLC plays a crucial role in the early
stage of drug development when information about the impurities and degradation
products in drug substance and drug product is inadequate.

Thin layer chromatography, or TLC, is a method for analyzing mixtures by

separating the compounds in the mixture. TLC can be used to help determine the
number of components in a mixture, the identity of compounds, and the purity of a
compound. By observing the appearance of a product or the disappearance of a
reactant, it can also be used to monitor the progress of a reaction. TLC is a sensitive
technique - microgram (0.000001 g) quantities can be analyzed by TLC - and it takes
little time for an analysis (about 5-10 minutes).

2.3 Principle:

TLC based upon Adsorption Principle

like other chromatographic techniques, thin-layer chromatography (TLC) depends on

the separation principle. The separation relies on the relative affinity of compounds
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towards both the phases. The compounds in the mobile phase move over the surface
of the stationary phase. The movement occurs in such a way that the compounds
which have a higher affinity to the stationary phase move slowly while the other
compounds travel fast. Therefore, the separation of the mixture is attained. On
completion of the separation process, the individual components from the mixture
appear as spots at respective levels on the plates. Their character and nature are
identified by suitable detection techniques.
Like Dissolves Like Concept in Chromatography:
A simple way to predict which compounds will dissolve in other compounds is the
phrase ‘like dissolves like’. What this means is that polar compounds dissolve polar
compounds, nonpolar compounds dissolve nonpolar compounds, but polar and
nonpolar do not dissolve in each other.

2.4 Plate Preparation:

TLC plates are usually commercially available, with standard particle size ranges to
improve reproducibility. They are prepared by mixing the adsorbent, such as silica
gel, with a small amount of inert binder like calcium sulfate (gypsum) and water.
This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass,
thick aluminum foil, or plastic. The resultant plate is dried and activated by heating
in an oven for thirty minutes at 110 °C. The thickness of the absorbent layer is
typically around 0.1–0.25 mm for analytical purposes and around 0.5–2.0 mm for
preparative TLC.

Stationary Phase:

 The most commonly used stationary phases are adsorbents e.g., fine powders of
silica gel G, alumina, Kieselguhr and cellulose, they are specially prepared for
TLC.
 Many of these are available with a fluorescent compound e.g. ZnS incorporated
inorder to facilitate the detection of the resolved components of the mixture when
viewed under UV light.

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 Silica gel G is slightly acidic whereas alumina is slightly basic but neutral
alumina is also available.
 Silica gel and alumina particles contain hydroxyl groups on their surface which
will form hydrogen bond with polar molecules.
 Alumina is preferred for the separation of weakly polar compounds, but silica gel
is preferred for polar compounds such as amino acid and sugars.
 Other absorbents suitable for special purpose are polyamides, magnesium
silicate, calcium silicate, activated charcoal, modified cellulose with ion
exchange properties and the various forms of organic gel e.g. Sephadex, Bio-Gel.
 Support used for the stationary phase in TLC is a glass plate, aluminium metal
plate or plastic strip.
 Glass plates can be used in a number of sizes ranging from microscope slides to
larger plates (20cm X 20cm X 20cm).
 Binder is essential for good adherence of the adsorbent to the plate.
 Commonly used binder is Gypsum (calcium sulphate) which is incorporated at a
level of 10-15%.
 Starch and certain organic polymers are also used as binders.

I Chromatography Stationary Phase Polarities

N Polymethyl siloxane
C
Methyl/Phenyl siloxane
R
E Cyanopropyl siloxane Carbowax (polyethylene glycol) *
A
Reverse Phase (hydrocarbon-coated silica e.g., C-18)
S
I Paper
N Cellulose
G
P Starch
O Calcium sulphate
L
A Silica (silica gel)
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R Florisil (magnesium silicate)
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I
T Magnesium oxide
y Alumina (aluminium oxide; acidic, basic or neutral) Activated carbon (charcoal,
 Mobile Phase

I
N
C
R
E
A
S
I
N
G
P
O
L
A
R
I
T
y

 The solvent used are listed below in order of increasing polarity, it is known as
eluotropic series.
 Petroleum ether < n-hexane < carbon tetrachloride < toluene < benzene < chloroform
< dichloromethane < diethyl ether < n-butanol < isopropanol < acetone < methanol <
water
 Movement of the solute increases with increasing solvent polarity.
 The solvent is employed in the form of a pool at the bottom of the developing
chamber.
 Usually the solvent or eluant is made to ascend on the plate, hence the name
ascending TLC.
 The solvent travels through the system by capillary action, thus solvent velocity is
determined by the nature and packing structure of the adsorbent.
 The saturation of the development chamber with solvent vapor has a significant
effect on solute migration.

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 Specifically, with under-saturation solvent and solute migrations are slow and not
smoothly ascending.
 This problem can be overcome by lining the developing chamber with a filter paper
which is then saturated with developing solvent to provide uniform chamber
saturation.
 Selection of the developing solvent is done by preliminary trial runs using
microscopic slides or small strips.

2.5 Procedure of Thin layer chromatography:

Step I: Plate preparation:
 Cleaning – Glass plates must be carefully cleaned with detergent to remove adhering
particles rinsed thoroughly with distilled water, placed in a metal rack and dried in an
oven.
 The plate should be handled by the edges or by the under-surface which is not to be
coated with the adsorbent.
 Failure to take the precaution and grease contamination on the glass surface may
result in the formation of poor quality mechanically unstable layer, which is liable to
be flaking.

Step II: Adsorbent selection:

 Commonly used adsorbents are silica gel, alumina, cellulose and polyamide.

Step III: Slurry preparation:

 Slurry is prepared by the slow addition with stirring of adsorbent, e.g., silica gel or
alumina, to a suitable solvent like water, dichloromethane in a wide mothed capped
bottle.
 Too thick or too thin slurries should be avoided.

Step IV: Coating of the TLC plate:

 The supporting plate (glass, metal etc.) should fulfil the following requirements:
 Uniform thickness
 Inert to solvent, solute, stationary phase, identification reagents, procedures

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 Sufficient strength to allow vertical development.
 Thin layer may be prepared by pouring, dipping, spraying or spreading the adsorbent
slurry over the plate.
 Generally, the best procedure for the preparation of uniform layers or films is the use
of the commercial spreader.
 However, the best source of uniform plates may be pre-coated plates that are
available from manufacturers and suppliers.

Step V: Activation of TLC plate:

 After the slurry has been spread out evenly the plates are placed horizontally to set
for approximately 10 minutes in a fume cupboard.
 The surplus adsorbent is removed from the glass edged by means of razor blade or
glass rod.
 The plate is then activated by heating at 110oC for 1 hour in an oven.
 The drying conditions may vary with the nature of the adsorbent, binder and the
solvent.
 After they are dry, the plates should be cooled to room temperature and stored in
desiccators until used.
 Cellulose and polyamide plates are allowed to dry at room temperature and are not
normally heated, then stored in a dust free cabinet.
 After drying, normal layer thickness is in the range of 150mm for analytical and
2mm for preparative TLC system.

Step VI: Sample preparation:

 The mixture (e.g., a mixture of amino acids) to be analysed is dissolved in a suitable
solvent (0.5-3%).
 The selected solvent should be volatile for rapid evaporation of the solvent is
desirable as this leads to the formation of a small-diameter spot which results in a
better separation during chromatographic development process.

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 Reference compounds are similarly prepared and applied to the adsorbent on the
same plate alongside the mixture spot, this helps more ready interpretation of the
chromatogram.

Step VII: Sample application on the TLC plate:

 Wipe any excess adsorbent from the back and edges of the plate.
 Sample should normally be applied about 5 mm (for small plates) to 10 mm (larger
plates) from the edge of the plate.
 However, care should be taken not to immerse the spot in the solvent pool in the
development chamber.
 The spots should be separated from each other by at least 10 mm for larger plates.
 Sample application is performed by spotting or streaking the thin layer.
 Analytical plates are usually spotted while preparative plates are streaked.

Step VIII: Spotting:

 Done with a melting point capillary tube or micropipette or microliter syringe.
 The applicator is charged by dipping the capillary end into the solution.
 The solution is then transferred by touching the tip of the capillary onto the adsorbent
layer.
 The sample volume is usually in the range of 1-10.
 The spot must be as small as possible for better separation and minimum tailing.

Step IX: Developing the chromatogram (Elution)

Tank selection:
 TLC can be developed in wide variety of chambers. For example, microscopic slides
are developed in a small cylindrical glass jar or wide mouthed screw capped glass
bottles.
 Larger plates (20cm X 20cm) require a rectangular glass tank of suitable dimension
with airtight lid.
Tank saturation:

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 The inside of the development tank is lined with filter paper leaving a gap for
viewing the chrom0plate.
 The filter paper is saturated with the selected developing solvent and the requisite
amount of the developing solvent is carefully poured down the side of the tank.
 Then the tank is closed with lid and allowed to stand for about 5 minutes so that the
atmosphere in the tank becomes saturated with solvent vapour.
Insertion of the plate:
 The loaded plate with the origin of spots or baseline towards the bottom of the tank is
inserted and tilted.
 Care should be taken that the solvent level is below the origin or baseline.
 The tank is recapped or closed and the solvent is allowed to ascend by capillary
action to the finishing line which is about 0.5 cm away from the top edge of the
adsorbent layer.
 The time required to complete this development varies greatly with the solvent
composition and the nature of the adsorbent.
 When the solvent reaches the finishing line, the position of the solvent is marked on
the absorbent layer and immediately the plate is removed from the development tank.
 After removal, plate is air dried.

Step X: Detection of spots/ Visualization of solutes:

 The position of coloured components can be seen in daylight without any difficulty.
 For colourless solutes, visualization or detection techniques are many, such as:
Iodine vapor:
 This is a semi-destructive general method for most organic compounds.
 The dried plate is allowed to stand in a closed tank containing iodine crystals
scattered over the tank bottom.
 The spots are revealed as brown stains.
 Their positions should be marked as soon as the plate has been removed from iodine
tank since standing in air for a short while causes the iodine to evaporate and the
stained spots to disappear.

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 A commonly used semi-destructive visualization method is to expose a developed
TLC plate to iodine (I2)(I2) vapor. An "iodine chamber" can be created by adding a
few iodine crystals to a TLC chamber, or by adding a few iodine crystals to a
chamber containing a portion of powdered silica or alumina (Figure 2.33a). When a
developed TLC plate is placed in the chamber and capped, the iodine sublimes and
reacts with the compounds on the plate, forming yellow-brown spots (Figure 2.33d).
The coloration occurs because iodine forms colored complexes with many organic
compounds. This stain will work with approximately half the compounds you may
encounter.

 This method is considered "semi-destructive" because complexation is reversible,

and the iodine will eventually evaporate from the TLC plate, leaving the original
compound behind. When the coloration fades, it is theoretically possible to use
another visualization technique on the TLC plate, although it's possible the
compound may have also evaporated by that time.

Procedure for visualization of TLC plate with iodine:

1. If not already prepared, make an iodine chamber in a fume hood, place a few
centimeters of powdered silica or alumina in a screw-capped TLC chamber and add a
few crystals of solid iodine. A beaker and watch glass will not work in this context as
the iodine vapours will escape. Let the silica or alumina and iodine sit together for a
while with periodic swirling, and eventually the powder will become orange from
adsorbing the iodine vapor.
2. In a fume hood, place the developed TLC plate in the iodine chamber with forceps
and close the lid. Gently shake the chamber to bury the TLC plate in the iodine-
stained silica or alumina until the spots become coloured. Many spots will appear
yellow-brown almost immediately, and may darken with extended time. For many
compounds, it takes less than 10 seconds to develop a plate, but some compounds
require 10 minutes or longer.
3. Promptly record appropriate observations of the TLC in your notebook, or circle the
spots with a pencil, as the colors will soon fade as the iodine evaporates from the
plate. Further visualization may be attempted after the colour fades.
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How Does Thin Layer Chromatography Work?

The stationary phase - silica gel G

Silica gel G is a form of silicon dioxide (silica). The silicon atoms are joined via
oxygen atoms in a giant covalent structure. However, at the surface of the silica gel
G, the silicon atoms are attached to -OH groups. So, at the surface of the silica gel G
you have Si-O-H bonds instead of Si-O-Si bonds. The diagram shows a small part of
the silica surface.

The surface of the silica gel G is very polar and, because of the -OH groups, can
form hydrogen bonds with suitable compounds around it as well as van der Waals
dispersion forces and dipole-dipole attractions.

The other commonly used stationary phase is alumina - aluminium oxide. The
aluminium atoms on the surface of this also have -OH groups attached. Anything we
say about silica gel G therefore applies equally to alumina.

What separates the compounds as a chromatogram develops?

As the solvent begins to soak up the plate, it first dissolves the compounds in the spot
that you have put on the base line. The compounds present will then tend to get
carried up the chromatography plate as the solvent continues to move [Link]
fast the compounds get carried up the plate depends on two things:
 How soluble the compound is in the solvent. This will depend on how much
attraction there is between the molecules of the compound and those of the
solvent.

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  How much the compound sticks to the stationary phase - the silica gel, for
example. This will depend on how much attraction there is between the
molecules of the compound and the silica gel G.

Suppose the original spot contained two compounds - one of which can form
hydrogen bonds, and one of which can only take part in weaker van der Waals
interactions. The one which can hydrogen bond will stick to the surface of the silica
gel more firmly than the other one. We say that one is adsorbed more strongly than
the other. Adsorption is the name given to one substance forming some sort of bonds
to the surface of another one.

Adsorption isn't permanent - there is a constant movement of a molecule between

being adsorbed onto the silica gel surface and going back into solution in the solvent.
Obviously the compound can only travel up the plate during the time that it is
dissolved in the solvent. While it is adsorbed on the silica gel, it is temporarily
stopped - the solvent is moving on without it. That means that the more strongly a
compound is adsorbed, the less distance it can travel up the plate.

In the example we started with, the compound which can hydrogen bond will adsorb
more strongly than the one dependent on van der Waals interactions, and so won't
travel so far up the plate.

TLC Diagrammatic view

Figure 2: TLC Diagrammatic View

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 Figure 3:TLC

Cyclohexane: Acetone (6:4)

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 Figure 4

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 3. UV visible Spectroscopy
3.1 What is UV Visible Spectroscopy?

UV-visible spectroscopy is an analytical technique that measures the number of

discrete wavelengths of UV or visible light that are absorbed by or transmitted
through a sample in comparison to a reference or blank sample. This property is
influenced by the sample composition, potentially providing information on what is
in the sample and at what concentration. Since this spectroscopy technique relies on
the use of light, let’s first consider the properties of light.
Light has a certain amount of energy which is inversely proportional to its
wavelength. Thus, shorter wavelengths of light carry more energy and longer
wavelengths carry less energy. A specific amount of energy is needed to promote
electrons in a substance to a higher energy state which we can detect as absorption.
Electrons in different bonding environments in a substance require a different
specific amount of energy to promote the electrons to a higher energy state. This is
why the absorption of light occurs for different wavelengths in different substances.
Humans are able to see a spectrum of visible light, from approximately 380 nm,
which we see as violet, to 780 nm, which we see as red. UV light has wavelengths
shorter than that of visible light to approximately 100 nm. Therefore, light can be
described by its wavelength, which can be useful in UV-visible spectroscopy to
analyze or identify different substances by locating the specific wavelengths
corresponding to maximum absorbance.
3.2 Principle of UV Spectroscopy
Basically, spectroscopy is related to the interaction of light with matter. As light is
absorbed by matter, the result is an increase in the energy content of the atoms or
molecules.
When ultraviolet radiations are absorbed, this results in the excitation of the electrons
from the ground state towards a higher energy state.

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Molecules containing π-electrons or nonbonding electrons (n-electrons) can absorb
energy in the form of ultraviolet light to excite these electrons to higher anti-bonding
molecular orbitals.
The more easily excited the electrons, the longer the wavelength of light they can
absorb. There are four possible types of transitions (π–π*, n–π*, σ–σ*, and n–σ*),
and they can be ordered as follows: σ–σ* > n–σ* > π–π* > n–π*
The absorption of ultraviolet light by a chemical compound will produce a distinct
spectrum that aids in the identification of the compound.

3.3 Instrumentation or Parts of UV Spectroscopy:

Light Source:
Tungsten filament lamps and Hydrogen-Deuterium lamps are the most widely used
and suitable light sources as they cover the whole UV region.
Tungsten filament lamps are rich in red radiations; more specifically they emit the
radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below
375 nm.
Monochromator:
Monochromators generally are composed of prisms and slits.
Most of the spectrophotometers are double beam spectrophotometers.
The various wavelengths of the light source which are separated by the prism are
then selected by the slits such the rotation of the prism results in a series of
continuously increasing wavelengths to pass through the slits for recording purposes.
The beam selected by the slit is monochromatic and further divided into two beams
with the help of another prism.
Sample and reference cells:
One of the two divided beams is passed through the sample solution and the second
beam is passé through the reference solution.
Both sample and reference solution is contained in the cells.
These cells are made of either silica or quartz. Glass can’t be used for the cells as it
also absorbs light in the UV region.

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Detector
Generally, two photocells serve the purpose of the detector in UV spectroscopy.
One of the photocells receives the beam from the sample cell and the second detector
receives the beam from the reference.
The intensity of the radiation from the reference cell is stronger than the beam of the
sample cell. This results in the generation of pulsating or alternating currents in the
photocells.
Amplifier:
The alternating current generated in the photocells is transferred to the amplifier.
The amplifier is coupled to a small servometer.
Generally, the current generated in the photocells is of very low intensity, the main
purpose of the amplifier is to amplify the signals many times so we can get clear and
recordable signals.
Recording devices:
Most of the time amplifier is coupled to a pen recorder which is connected to the
computer.
The computer stores all the data generated and produces the spectrum of the desired
compound.

3.4 Applications of UV Spectroscopy:

Detection of Impurities:
It is one of the best methods for the determination of impurities in organic
molecules.
Additional peaks can be observed due to impurities in the sample and it can be
compared with that of standard raw material.
By also measuring the absorbance at a specific wavelength, the impurities can be
detected.

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 Ethanol

Benzil and
Phenytoin and
Ethanol
Figure 5: Instrumentation of UV

3.5 Monitoring Reaction of Phenytoin:

Reaction is monitored by checking their absorbance

Firstly, we take the baseline in UV visible Spectroscopy by using the Ethanol

Then we take the Benzil as a starting material and take absorbance in UV using 10
ppm solution of Benzil + Ethanol as shown in Figure 5

After that we take the absorbance of Phenytoin (Final Product) using the 10 ppm
Solution of Phenytoin dissolved in Ethanol as Shown in Figure 5

Then we compare the absorbance spectra of both starting material and final product.
If the Absorbance Peak of two Compounds (Starting material And Final Product) is
Collapse on each other than the starting material is present in Final product

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In this case the Absorbance Peak are not Collapse on each other and Separate Peak
was Observed then it is conformed that the Phenytoin is pure or No Staring Material
is Present in it

Figure 6: UV Spectra of Benzil & Phenytoin

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 Figure 7: UV Spectra of Benzil & Phenytoin

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 4. Bibliography:
1. Vogel's Textbook of Practical Organic Chemistry by Brian S. Furniss,
Antony J. Hannaford, Peter W. G. Smith & Austin R. Tatchell; Fifth Edition;
Page No. 1153.

2. Practical in organic chemistry, by Hitesh G. Raval, Sunil L. Baldania and

Dimal A. Shah, Nirav Prakashan, Page No. 313.

3. Instrumental methods of Chemical Analysis. by Gurdeep R. Chatwal & Sham

R· Anand Himalaya Publishing House Page No- 2.107-2148.

4. [Link]

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