(CHE(O) 510

DISSERTATION
REPORT

1
Roll No. : 32 Exam No. :

PAPER No. : CHO (510)

PAPER NAME: Dissertation
A DISSERTATION REPORT

"ASSAY OF CLOZAPIN ORDISPERSIBLE TABLETS"

SUBMITTED
TO
THE GUJARAT UNIVERSITY

IN PARTIAL FULLFILLMENT OF THE

REQUIREMENT FOR THE DEGREE OF

MASTER OF SCIENCE
IN
CHEMISTRY
BY
THUMMAR DHAVAL BHAGAVANBHAI

DEPARTMENT OF CHEMISTRY
GUJARAT ARTS AND SCIENCE COLLEGE
ELLIS BRIDGE,
AHMEDABAD-380006
GUJARAT, INDIA
APRIL 2022

2
 DECLARATION

I hereby, declare that the dissertation report of CHE (O) 510 “ASSAY OF CLOZAPINE
ORDISPERSIBLE TABLETS” submitted for the in partial fulfillment of the requirement for
degree of Master of Science. The dissertation work is carried out by me at Global Analytical
Laboratory.

Date: Name: Thummar Dhaval

Place: Ahmedabad Roll no: 32
Exam No.:

3
4
 ACKNOWLEDGEMENT

At the time of presenting this report it is necessary that I would thank all the
persons who helped us directly or indirectly and gave me proper guideline.

The January internship at Global Analytical Laboratory, Ahmedabad has been

a truly enriching and highly enjoyable experience with a great value addition
to my knowledge, which will be surely very helpful in future carrier.

I take this opportunity to thank all those people who helped in making this
experience a memorable one. First of all, I would like to sincerely thank to
Global Analytical Laboratory for giving me this opportunity.

It was the esteemed guidance of my project guide Prof. R.R. Patel

That brought this project to successful completion. I express my sincere
gratitude to all the members of Global Analytical Laboratory
Who took out some time from their busy schedule and shared their valuable
knowledge and experience of the organization.

5
 PREFACE

Any amount of theoretical knowledge is incomplete without exposure to

Industrial practice. Practical knowledge means visualization and application
of knowledge which we read in books.

In addition of technical knowledge, here I got a chance to improve skill like

self-esteem. Teamwork, creative thinking, communication skill, etc. which
are very important for making the career.

Theoretical studies cannot be perfect without practical training. Hence in‐lab

training is of great importance for a chemistry student. Teaching gives
theoretical aspects of chemistry, but practical training gives knowledge of
industrial activities.

My aim for this internship at Global Analytical Laboratory, Ahmedabad was

to get a detail idea of different aspects of Quality control department with
different services and analysis their performance and apply
my already acquired knowledge to perform various reactions to them.
Besides the chemical aspects, I also tried to observe important aspects of
Laboratory behavior, discipline and safety precautions.

Thus, training report presents a detailed summary of my enriching

experience at the Global Analytical Laboratory

DHAVAL THUMMAR

6
 Index

Sr. No Title Pg. No

1. CHROMATOGRAPHY 8

2. HPLC 8

3. Parameters 18

4. HPLC Analysis 20

5. Experimental Work: - Assay of 23

Clozapine Ordispersibe Tablets

6. Chromatogram 27

7. CONCLUSION 33

8. REFFERNCES 34

7
 1. CHROMATOGRAPHY
It is defined as, it is analytical method in which separation of active
constituent in complex mixture, and the mixture was distributed in two phases i.e.,
stationary phase and mobile phase is known as chromatography.

It is technique is used for separation, purification, Identification and extraction of

compound. It is method it can consist of two phases;
(a) Stationary phase is constant phase or column packaging material.
(b) Mobile phase is moveable phase.

The basic principle of chromatography is based on Adsorption and partition

chromatography.

Adsorption chromatography - The affinity of molecules towards stationary phase is

known as Adsorption chromatography.

Partition chromatography - The molecule can move in two phases of liquid is known
as partition chromatography. It is important for qualitative and quantitative analysis.
[1].

2. HPLC

 Basics of HPLC System:

HPLC, also called high performance liquid chromatography or high-pressure

liquid chromatography is an analyzer used for identifying, quantifying,
separating and purifying chemical compounds.

8
 Configuration of an HPLC system:

An HPLC system consists of a pumping unit, sample-injection unit, separation

unit, detection unit and data processing unit. Each of these unit is essential for
performing the analysis.

9
 Principle of Liquid Chromatography:
High-performance liquid chromatography (HPLC; formerly referred to as high-
pressure liquid chromatography), is a technique in analytical chemistry used to
separate, identify, and quantify each component in a mixture. It relies on pumps
to pass a pressurized liquid solvent containing the sample mixture through a
column filled with a solid adsorbent material. Each component in the sample
interacts slightly differently with the adsorbent material, causing different flow
rates for the different components and leading to the separation of the
components as they flow out the column.

HPLC has been used for medical (e.g., detecting vitamin D levels in blood serum),
legal (e.g., detecting performance enhancement drugs in urine), research (e.g.,
separating the components of a complex biological sample, or of similar synthetic
chemicals from each other), and manufacturing (e.g., during the production
process of pharmaceutical and biological products) purposes

Chromatography can be described as a mass transfer process involving

adsorption. HPLC relies on pumps to pass a pressurized liquid and a sample
mixture through a column filled with a sorbent, leading to the separation of the
sample components. The active component of the column, the sorbent, is
typically a granular material made of solid particles (e.g., silica, polymers, etc.), 2–
50 micrometers in size.

The components of the sample mixture are separated from each other due to
their different degrees of interaction with the sorbent particles. The pressurized
liquid is typically a mixture of solvents (e.g., water, acetonitrile and/or methanol)
and is referred to as a "mobile phase". Its composition and temperature play a
major role in the separation process by influencing the interactions taking place
between sample components and sorbent.

These interactions are physical in nature, such as hydrophobic (dispersive),

dipole–dipole and ionic, most often a combination.

HPLC is distinguished from traditional ("low pressure") liquid chromatography

because operational pressures are significantly higher (50–350 bar), while
ordinary liquid chromatography typically relies on the force of gravity to pass the
mobile phase through the column.
10
 Schematic representation of an HPLC system

The schematic of an HPLC instrument typically includes a sampler, pumps, and

a detector. The sampler brings the sample mixture into the mobile phase stream
which carries it into the column. The pumps deliver the desired flow and
composition of the mobile phase through the column.

The detector generates a signal proportional to the amount of sample

component emerging from the column, hence allowing for quantitative analysis
of the sample components. A digital microprocessor and user software control
the HPLC instrument and provide data analysis. Some models of mechanical
pumps in a HPLC instrument can mix multiple solvents together in ratios changing
in time, generating a composition gradient in the mobile phase.

11
 a) Mobile Phase:

The mobile phase is composed of one or more reagents that circulate through
the HPLC at a preset flow rate and ratio. Reagents are stored in mobile phase
reservoirs and drawn out through a tube by the pump. Depending on the
nature of the samples, an isocratic or gradient mobile phase can be used. An
isocratic phase maintains a consistent solvent concentration throughout the
analysis and requires one solution reservoir. A gradient elution varies the
concentration of mobile phase based on a preset schedule.

b) Injector:

The injector draws a preset volume of liquid from the sample vial and injects it
into the mobile phase flowing through the system. Depending on the
chemical response needed and the amount of samples available, injection
volumes typically range from 10 to 50 ml. Most modern HPLCs are equipped
with an automated injector located within a temperature-controlled
enclosure.

c) Pump:

The pump is responsible for circulating the mobile phase and sample through
the HPLC system. It runs at a preset speed that determines the system’s
pressure. Different types of pumps can be used, with the majority pumping at
pressures under 400 bar. Important pieces of the pump include the motor,
piston, seal, check valves and purge valves to direct solvent to the waste.

Pumps vary in pressure capacity, but their performance is measured on their

ability to yield a consistent and reproducible flow rate. Pressure may reach as
high as 60 MPa (6000 lbf/in2), or about 600 atmospheres. Modern HPLC
systems have been improved to work at much higher pressures, and therefore
are able to use much smaller particle sizes in the columns.

12
 d) Column:

The column is the HPLC’s stationary phase. It is responsible for separating the
individual molecules within each sample before they are characterized by the
detector. The column is packed with material that is designed to selectively
interact with specific molecules. The retention time of molecules by the
stationary phase depends on their affinity for the stationary compared to that
of the mobile phases. Depending on the analysis, reverse phase or normal
phase columns can be used.

HPLC columns are made with smaller sorbent particles (2–50 micrometer in
average particle size). This gives HPLC superior resolving power (the ability to
distinguish between compounds) when separating mixtures, which makes it a
popular chromatographic technique.

The components of the sample move through the column at different

velocities, which are function of specific physical interactions with the sorbent
(Also called stationary phase). The velocity of each component depends on its
chemical nature, on the nature of the stationary phase (column) and on the
composition of the mobile phase.

13
 Types of HPLC columns:

 Normal Phase Columns:

Normal phase columns are used when the stationary phase is more polar than
the mobile phase.

In normal phase chromatography, the polar stationary phase (also known as

packing material) could be silica gel and the less polar mobile phase could be
hexane, or another organic solvent.

This type of column is commonly used for samples with small molecules, like
organic acids or pharmaceuticals. It can also be used for biomolecules, such as
glycosylated proteins.

 Reverse Phase Columns:

Reverse phase columns are used in reverse phase chromatography, when the
stationary phase is less polar than the mobile phase. In other words, the
revers of normal phase chromatography.

Water is often used as the mobile phase and common stationary phases are
acetonitrile, methanol, and tetrahydrofuran (THF).

This method is more widely used than normal phase chromatography, as it can
be used for a wide range of analytical applications.

 Ion Exchange Columns:

Column separation is slightly different in ion exchange chromatography. Instead
of relying on polarity alone, this method uses charge to separate substances
that can be easily ionized.

14
 Typically, the stationary phase is an acid with either a positive or negative
charge and the mobile phase is a polar aqueous buffer, like salt water.

In this type of column, separation occurs due to the attractive ionic forces
between the molecules in the sample and the charged stationary phase.

This method is usually used to separate carbohydrates, amino acids and

proteins.

 Size Exclusion Columns:

Size exclusion chromatography separates the sample using particle size.

The stationary phase needs to have porous particles for size exclusion
chromatography so molecular sieves (such as zeolites), polysaccharides
and polymers (like a typical silica column) are most commonly used.

This technique is used to separate proteins and carbohydrates.

e) Detector:

The detector is the component that identifies the absorbency or fluorescence

profile of each molecule flowing through the system. Depending on the
molecules being analyzed, ultraviolet, visible or fluorescence detectors can be
used. The detection wavelength is preset before the analysis and the resulting
data used to create a spectral profile for each chemical detected.

When selecting a detector, it is important to consider the sample type being

analyzed. Some sample types will not work or will have lower sensitivity on
particular detectors. Also of consideration is the type of information that is
being sought by the analysis and what method will be used when running the
column, for example, some detectors will work with the gradient method
while others will not.

15
1. HPLC Conductivity Detector 2.HPLC Evaporative Light Scattering Detector

3.HPLC Fluorescence Detector 4. HPLC Refractive Index Detector

5.HPLC UV VIS Detector

16
 f) Isocratic And Gradient Elution

A separation in which the mobile phase composition remains constant

throughout the procedure is termed isocratic (meaning constant composition).
The word was coined by Csaba Horvath who was one of the pioneers of HPLC.,
The mobile phase composition does not have to remain constant.

A separation in which the mobile phase composition is changed during the

separation process is described as a gradient elution. One example is a gradient
starting at 10% methanol and ending at 90% methanol after 20 minutes. The two
components of the mobile phase are typically termed "A" and "B";

Term A is the "weak" solvent which allows the solute to elute only slowly,
Term B is the "strong" solvent which rapidly elutes the solutes from the
column.

In reversed-phase chromatography, solvent A is often water or an aqueous

buffer, while B is an organic solvent miscible with water, such as acetonitrile,
methanol, THF, or isopropanol.

In isocratic elution, peak width increases with retention time linearly according
to the equation for N (number of theoretical plates). This leads to the
disadvantage that late-eluting peaks get very flat and broad. Their shape and
width may keep them from being recognized as peaks. Gradient elution decreases
the retention of the later-eluting components so that they elute faster, giving
narrower (and taller) peaks for most components. This also improves the peak
shape for tailed peaks, as the increasing concentration of the organic eluent
pushes the tailing part of a peak forward. This also increases the peak height (the
peak looks "sharper"), which is important in trace analysis.

The gradient program may include sudden "step" increases in the percentage of
the organic component, or different slopes at different times – all according to
the desire for optimum separation in minimum time. In isocratic elution, the
selectivity does not change if the column dimensions (length and inner diameter)
change – that is, the peaks elute in the same order. In gradient elution, the elution
order may change as the dimensions or flow rate change. The driving force in
reversed phase chromatography originates in the high order of the water

17
structure. The role of the organic component of the mobile phase is to reduce this
high order and thus reduce the retarding strength of the aqueous component.

3. Parameters

 Theoretical

HPLC separations have theoretical parameters and equations to describe the

separation of components into signal peaks when detected by instrumentation
such as by a UV detector or a mass spectrometer.

The parameters are largely derived from two sets of chromatographic theory:
(1) Plate theory (as part of Partition chromatography),
(2) Rate theory (chromatography / Van Demeter equation).

Of course, they can be put in practice through analysis of HPLC chromatograms,

although rate theory is considered the more accurate theory. They are analogous
to the calculation of retention factor for a paper chromatography separation, but
describes how well HPLC separates a mixture into two or more components that
are detected as peaks (bands) on a chromatogram.

The HPLC parameters are the:

 Efficiency factor (N),
 Retention factor (kappa prime),
 Separation factor (alpha)

Together the factors are variables in a resolution equation, which describes

how well two components' peaks separated or overlapped each other. These
parameters are mostly only used for describing HPLC reversed phase and HPLC
normal phase separations, since those separations tend to be more subtle than
other HPLC modes (e.g., ion exchange and size exclusion).

Void volume is the amount of space in a column that is occupied by

solvent. It is the space within the column that is outside of the column's
internal packing material. Void volume is measured on a chromatogram as
the first component peak detected, which is usually the solvent that was
present in the sample mixture; ideally the sample solvent flows through the
18
column without interacting with the column, but is still detectable as distinct
from the HPLC solvent. The void volume is used as a correction factor.

(a) Efficiency factor (N) practically measures how sharp component peaks on
the chromatogram are, as ratio of the component peak's area ("retention
time") relative to the width of the peaks at their widest point (at the
baseline). Peaks that are tall, sharp, and relatively narrow indicate that
separation method efficiently removed a component from a mixture; high
efficiency. Efficiency is very dependent upon the HPLC column and the HPLC
method used. Efficiency factor is synonymous with plate number, and the
'number of theoretical plates'.

(b) Retention factor (kappa prime) measures how long a component of the
mixture stuck to the column, measured by the area under the curve of its
peak in a chromatogram (since HPLC chromatograms are a function of
time). Each chromatogram peak will have its own retention factor (e.g.,
kappa1 for the retention factor of the first peak). This factor may be
corrected for by the void volume of the column.

(c) Separation factor (alpha) is a relative comparison on how well two

neighboring components of the mixture were separated (i.e., two
neighboring bands on a chromatogram). This factor is defined in terms of a
ratio of the retention factors of a pair of neighboring chromatogram peaks,
and may also be corrected for by the void volume of the column. The
greater the separation factor value is over 1.0, the better the separation,
until about 2.0 beyond which an HPLC method is probably not needed for
separation. Resolution equations relate the three factors such that high
efficiency and separation factors improve the resolution of component
peaks in a HPLC separation

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4. HPLC Analysis

 Overview of Method Validation

The search for the reliable range of a method and continuous application of this
knowledge is called validation. It can also be defined as the process of
documenting that the method under consideration is suitable for its intended
purpose. Method validation involves all the procedures required to demonstrate
that a particular method for quantitative determination of an analyte is reliable
for the intended application. Validation is also a proof of the repeatability,
specificity and suitability of the method. Validation is required to demonstrate the
performance of the method and reliability of analytical results. There are eight
different validation parameters as shown below:

 Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of

components which may be expected to be present. Typically these might include
impurities, degradants, matrix, etc.

 Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement

between the value which is accepted either as a conventional true value or an
accepted reference value and the value found.

 Precision

The precision of an analytical procedure expresses the closeness of agreement

(degree of scatter) between a series of measurements obtained from multiple
sampling of the same homogeneous sample under the prescribed conditions.
Precision may be considered at three levels: repeatability, intermediate precision
and reproducibility.

 Detection limit

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The detection limit of an individual analytical procedure is the lowest amount of
analyte in a sample which can be detected but not necessarily quantitated as an
exact value.

 Quantitation limit

The quantitation limit of an individual analytical procedure is the lowest amount

of analyte in a sample which can be quantitatively determined with suitable
precision and accuracy. The quantitation limit is a parameter of quantitative
assays for low levels of compounds in sample matrices, and is used particularly for
the determination of impurities and/or degradation products.

 Linearity

The linearity of an analytical procedure is its ability (within a given range) to obtain
test results which are directly proportional to the concentration (amount) of
analyte in the sample

 Range

The range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including these concentrations)
for which it has been demonstrated that the analytical procedure has a suitable
level of precision, accuracy and linearity.

 Robustness

The robustness of an analytical procedure is a measure of its capacity to remain

unaffected by small, but deliberate variations in method parameters and provides
an indication of its reliability during normal usage.

 System Suitability &Quantitation

System suitability tests are an integral part of gas and liquid chromatographic
methods. These tests are used to verify that the chromatographic system is
adequate for the intended analysis

Factors that may affect chromatographic behavior include the following:

21
  Composition, ionic strength, temperature, and apparent Ph of the mobile
phase
 Flow rate, column dimensions, column temperature, and pressure
 Stationary phase characteristics

The resolution, RS, is a function of the number of theoretical plates, N (also

referred to as efficiency), the separation factor, and the capacity factor, k. For a
given stationary phase and mobile phase, N may be specified to ensure that
closely eluting compounds are resolved from each other, to establish the general
resolving power of the system, and to ensure that internal standards are resolved
from the drug. This is a less reliable means to ensure resolution than is direct
measurement. Column efficiency is, in part, a reflection of peak sharpness, which
is important for the detection of trace components.

Replicate injections of a standard preparation or other standard solutions are

compared to ascertain whether requirements for precision are met. Unless
otherwise specified in the individual monograph, data from five replicate
injections of the analyte are used to calculate the relative standard deviation, %
RSD, if the requirement is 2.0% or less; data from six replicate injections are used
if the relative standard deviation requirement is more than 2.0 %

22
5. Experimental Work: - Assay Of Clozapine
Ordispersibe Tablets:

 INTRODUCTION
Clozapine is a tricyclic benzodiazepine, classified as an atypical antipsychotic
agent. It binds several types of central nervous system receptors and displays a
unique pharmacological profile. Clozapine is a serotonin antagonist, with strong
binding to 5-HT 2A/2C receptor subtype. It also displays a strong affinity to
several dopaminergic receptors but shows only weak antagonism at the
dopamine D2 receptor, a receptor commonly thought to modulate neuroleptic
activity. Agranulocytosis is a major adverse effect associated with the
administration of this agent.

Structure of clozapine
 Apparatus

Volumetric Flask: 5, 10, 50 mL

Pipette: 5, 10 mL
Graduated Cylinder: 1000 mL
Beaker: 100 and 50 mL
Laboratory Balance: Readability 220 gm
Analytical Balance: Readability 0.1 mg
Micro pipette: 1 mL
Column: Chrome Budget 100-5-C18 (150 mm×4.6 mm) 5µ
Column Receiver Date: 01-04-2023
Detector: Photo Diode Array
Wave lengths: 257 nm excitation filter.

23
  Reagent and Sample Preparation

Mobile phases: Prepare a mixture of 800 volumes of Methanol, 200 volumes of

water and 0.75 volumes of Triethylamine. Sonicate to degas before use.

Diluent: Prepare a mixture of 60 volumes of Methanol and 40 volumes of Water.

Sonicate to degas before use.

Standard preparation: Transfer an accurately weighed quantity about 31.3 mg of

Clozapine working standard into 50 ml volumetric flask, add about 25 ml of diluent
and sonicate to dissolve. Cool and dilute to volume with diluent and mix. Dilute
10 ml of this solution to 50 m with diluent and mix. (Set-1). (0.125 mg/mL of
Clozapine)

Note: Prepare Set-2 for Standard preparation as mentioned above.

Assay preparation: Transfer an accurately weighed 5 intact tablets into 50 ml

volumetric flask. Add 30 ml of methanol and sonicate to disperse the tablets.
Further sonicate for 15 minutes with intermittent shaking. (During sonication
necessary care should be taken to control the temperature between 20°C to
25°C). Cool and dilute to volume with methanol and mix. Filter the solution
through 0.45 u nylon filter and discard first few ml of filtrate.
Dilute 5 ml of this filtrate to 200 ml with diluent and mix. (0.125 mg/ml of
Clozapine)

 CHROMATOGRAPHIC SYSTEM:

System suitability: Equilibrate the column with mobile phase at prescribed

condition until a stable baseline is achieved. Separately inject single injection of
Diluent, six replicate injection of standard preparation Set-1 and single injection
of standard preparation Set-2 into liquid chromatography and record the
chromatograms.

In the chromatograms obtained with standard preparation Set-1

Relative standard deviation of replicate injections for area of the Clozapine peak
should not be more than 2.00%.

Tailing factor for the Clozapine peak should be between 0.80 to 2.00.
24
Theoretical plates (by tangent method) for Clozapine peak should not
be less than 1000.

Chromatographic parameters:
Column: Chrombudget 100-3-C18 (150 mm & 4.0 mm) 5 µ, Make: Bischoit or
equivalent
Flow rate: 1.0 mL/min
Wavelength: 237 nm
Injection Volume: 10 L.
Temperature:25°C
Retention Time: About 4.5 minutes for Clozapine peak
Run Time: 8.0 minutes

 Instrument Setup

Instrument Id: EQ-033 Series

Instrument Method: CLOZ_FP_AS_EQ-033
Software: E2 Chrome Elite
Version: 3.3.2SP2
Method: EQ-033\Routine Sample\SKIZ FP_ AS 090123. seg
Instrument Status: ON
Pump: Gradient

 Gradient Elution

Time Mobile Phase Mobile Phase

(min) A B
0 55 45
5 55 45
8 52 48
12 35 65
16 20 80
22 0 100
25 55 45
30 55 45

 Calculation

Assay as such basis = Mean Sample Area  Std wt. 2  100  50  Std. Potency
Mean Std. Area  100  50  Sample Wt.  2

25
26
6. Chromatogram

 Blank Solution

 Standard Solution set-1

27
28
29
 Table

 Standard Solution set- 2

30
 Sample Solution

31
32
 7. CONCLUSION:

• HPLC stands for High Performance Liquid Chromatography, and is a technique

used to separate different constituents of a compound using high pressure to
push solvents through the column. It is the most widely used technique to
identify, quantify and separate components of a mixture. HPLC can be used in
both qualitative and quantitative applications that are for both compound
quantification and identification. Normal phase HPLC is rarely used now, almost
all HPLC separation can be performed in reverse phase. Reverse phase HPLC
(RPLC) is ineffective in for only a few separation types. HPLC is applied for
molecular weight determination, in analytical chemistry, pharmaceutical and drug
science, clinical sciences, food technology, and consumer products, combinatorial
chemistry, polymer chemistry, environmental chemistry and green chemistry.

• A new HPLC method has been developed for the identification and quantification
of Clozapine. Low cost, faster speed, and satisfactory precision and accuracy are
the main features of this method. Method was successfully validated as per ICH
(International Council for Harmonisation) guidelines and statistical analysis proves that
method is sensitive, specific, and repeatable. It can be conveniently employed for
routine quality control analysis of Clozapine as bulk drug in marketed tablets.

33
 8. REFFERNCES:
1. https://www.khanacademy.org/science/chemistry/india

2. https://microbenotes.com/high-performance-liquid-chromatography-hplc/

3. https://www.linkedin.com/pulse/four-types-hplc-columns-how-many-do-you-know-rachel-zhang?trk=pulse-
article_more-articles_related-content-card

4. https://www.lcservicesltd.co.uk/

5. https://go.drugbank.com/drugs/DB00363

6. https://www.youtube.com/watch?v=C5849dE-zGE

34
THANK YOU

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