MODULE - 1:

UV-VISIBLE SPECTROPHOTOMETRY:

INTRODUCTION:
Ultraviolet-visible spectrophotometry is a technique used to measure light
absorbance across the ultraviolet and visible ranges of the electromagnetic
spectrum. When incident light strikes matter it can either be absorbed, reflected, or
transmitted. The wavelength range of UV radiation is 200 nm- 400 nm. There are
mainly two types of UV regions. 1. 200 nm to 400 nm that is called near ultraviolet
region. 2. Below 200 nm that is called far ultraviolet region. The wavelength of visible
radiation is 400 nm- 800 nm. Wavelength in UV and visible region is expressed in
nanometers or in angstroms. Absorption is expressed in terms of wave number
(cm-1).

The UV visible spectrophotometer is able to determine the concentration of specific

analytes in a microvolume by controlling the analysis of wavelengths and the
pathlength. The principle in UV visible spectrophotometer is based on Beer-Lambert
law. According to Beer- Lambert Law, the amount of light absorbed is directly
proportional to the concentration of the sample and the distance the light travels
through the sample; the pathlength.

PRINCIPLE:
● When matter absorbs ultraviolet radiation, the electrons present in it undergo
excitation. This causes them to jump from a ground state (an energy state
with a relatively small amount of energy associated with it) to an excited state
(an energy state with a relatively large amount of energy associated with it).
Four possible types of interactions or transitions can be observed. They are
(n-π, n-π*, σ- σ*, and n- σ*,). The order of the interaction according to the
energy is as follows: σ-σ*>n-σ*>π-π*>n-π*.

1
 ● σ-σ transition is a transition that needs larger energy to take place. An
example is methane, having a single bond C-H. and exhibits an absorption
maximum at 125 nm.
● the n-σ transition requires less energy than the o-o transition. Absorption
peaks occur below 200 nm, Saturated compounds having atoms with
unshared electrons undergo this transition. Examples are water and ethanol.
● π-π* and n-π* transitions require unsaturated functional groups to occur.
Alkene and alkynes are examples of π-π* transition and carbonyl compounds
are examples of n-π* transition.

INSTRUMENTATION:

The basic instrumentation of UV-visible spectrophotometer comprises of

1. Light source
2. Wavelength selector
3. Sample container
4. Detector

1) Light source: There are different sources of light in the UV spectrophotometer.

They are
a. Deuterium lamp
b. Hydrogen lamp
c. Tungsten lamp
d. Xenon discharge lamp
e. Mercury arc lamp

2) Wavelength selector: There are two types of wavelength selectors.

a. Filters: They are used to permit a certain band of wavelength. The simplest type
of filter is absorption filter.

b. Monochromators: It is an optical device that is used to select a narrow band of a

wavelength of light. It may be a quartz prism or grating. The Components of
a monochromator are
i. Slit
ii. Mirror
iii. Lens
iv. Grating/Prism

3) Sample containers: These are also called Cuvettes. The most common cuvette
size is 1cm. They are made of Quartz, Borosilicate and Plastic.
● Only quartz is transparent in both UV and Visible regions.
● Glass and plastic are suitable for the visible region only.
● Plastic cells are not used for organic solvents.

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 ● Glass is not suitable for UV region because it absorbs UV radiation.

4) Detectors: These are devices that indicate the existence of some physical
phenomenon. They are

a) photovoltaic cells or barrier- layer cell: It is also known as barrier layer or

photronic cell. It consists of a metallic base plate like iron or aluminium which acts as
one electrode.

b) phototubes or photoemissive tubes: It consists of an evacuated glass bulb.

There is a light sensitive cathode inside it in which the inner surface of the cathode is
coated with light sensitive layers such as potassium oxide and silver oxide.

e) photomultiplier tubes: It is a commonly used detector in UV spectroscopy. It

consists of a photo emissive cathode (a cathode which emits electrons when struck
by photons of radiation), several dynodes (which emit several electrons for each
electron striking them) and an anode.

APPLICATIONS:

1. It is useful in the structure elucidation of organic molecules, such as in detecting

the presence or absence of unsaturation, the presence of heteroatoms.

2. UV absorption spectroscopy can be used for the quantitative determination of 3.

compounds that absorb UV radiation.

This technique is used to detect the presence or absence of a functional group in the
compound.

4. Molecular weights of compounds can be measured spectrophotometrically by

preparing the suitable derivatives of these compounds.

3
 CALIBRATION OF UV-VISIBLE SPECTROPHOTOMETER:

AIM: To calibrate the UV-visible spectrophotometer by studying all the parameters in

it.
1. Control of Wavelength

2. Control of Absorbance

3. Photometric accuracy

4. Limit of Stray light

5. Resolution power

1. Control of Wavelength:

Procedure:

● Take a pair of matched quartz cells having a path length of 1 cm.

● Perform the base line correction with 1.4 M Perchloric acid solution from 200
to 600 nm.
● Remove the cell from the sample compartment.
● Rinse the sample cell with Holmium perchlorate solution, fill the cell with the
same, clean the smooth surfaces of the cell with tissue paper and place it in
the sample compartment.
● After scanning, go to operations and check for peaks and record the peaks.

Acceptance criteria:

Wavelength Maximum tolerance

241.15nm 240.15 to 242.15nm

287.15nm 286.15 to 288.15nm

361.50nm 360.50 to 362.50mm

536.30nm 533.30 to 539.30nm

2.0 Control of absorbance:

Procedure:

● Take a pair of matched quartz cells having a path length of 1 cm.

4
 ● Perform base line correction with 0.005 M Sulphuric acid from 200 to 400 nm.
● Remove the cell from the sample compartment.
● Rinse the sample cell with potassium dichromate UV solution, fill the cell with
the same, clean the smooth surfaces with tissue paper.
● Place the cell in sample compartment, scan from 200 to 400 nm and
measure
● the absorbances at 235 nm, 257 nm, 313 nm and 350 nm. Calculate the

specific absorbance using the following equation.

Specific absorbance at X nm =Absorbance at X nm

Absorbance at X nm Concentration of Potassium dichromate

• The specific absorbances against each wavelength should be as follows:

Wave length Specific absorbance Maximum tolerance

235 124.5 122.9 to 126.2

257 144.0 142.8 to 145.7

313 48.6 47.02 to 50.3

350 106.6 104.9 to 108.2

430 15.9 15.7 to 16.1

3.0 Photometric accuracy:

Procedure:

● Measure the absorbances of the above solutions at about 302 nm against

water blank.
● Check the absorbance of the above solutions at 302 nm using milli Q water as
blank.
● Record the absorbance at each wavelength in the Calibration Data Sheet.
● Check the Absorbance for each solution against the acceptance criteria given
below:

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Acceptance Criteria:

Solutions%w/v Absorbance at 302nm Acceptance criteria +/-

0.010

1.065 0.751 0.741 to 0.761

0.710 0.500 0.490 to 0.510

0.355 0.250 0.240 to 0.260

4.0 LIMIT OF STRAY LIGHT:

Procedure:

● Take a pair of matched quartz cells having a path length of 1 cm.

● Perform the base line correction with water.
● Remove the cell from the sample compartment.
● Rinse the cell in the potassium chloride solution, fill the cell with the same,
clean the smooth surfaces with tissue paper and place it in a sample
compartment.
● Measure the absorbance at 200 nm.

Acceptance Criteria: The absorbance should be more than 2.

5.0 RESOLUTION POWER:

Procedure:

● Record the spectrum in the range 260 to 275 nm of 0.020 % v/v toluene in
Hexane using Hexane in the reference cell.
● Calculate the ratio of the absorbance at the maximum at about 269 nm to that
at the minimum at about 266 nm.
● Acceptance Criteria: Ratio is not less than 1.5.

6
 DRUG PROFILE OF PARACETAMOL:

● Category: Analgesic and antipyretic

● Trade name: Tylenol, panadol.
● Molecular formula: CaH NO2
● Molecular weight:151.2 Melting point: 169.0° to 170.5°
● Solubility:
A) Very slightly soluble in cold water, considerably more soluble in hot water.
B) soluble in ethanol, methanol, dimethylformamide, ethylene dichloride,
acetone, and ethyl acetate.
C) very slightly soluble in chloroform
D) slightly soluble in ether
E) practically insoluble in petroleum ether, pentane, and benzene.
● Pka: 9.5 (25°).
● Partition coefficient: Log P(octanol/water), 0.5.
● Melting point: 169*c
● Appearance: White crystals or crystalline powder.
● Route of administration: orally, IV, rectal
● Onset of action: within 8 minutes by IV route and 30 minutes by mouth.
● Bioavailability: 60-80%
● Metabolism: predominantly in the liver
● Elimination t1/2: 1.9 to 2.5 hours
● Excretion: urine
● Uses: fever, pains, headache.
● Adverse effects: Liver toxicity or hepatotoxicity, loss of enzyme glucose 6
phosphate dehydrogenase.
● Side effects: nausea, vomiting, loss of appetite.

7
 ESTIMATION OF PARACETAMOL BY
UV - VISIBLE SPECTROSCOPY:
AIM
To estimate the Paracetamol in pure and pharmaceutical dosage form by
UV-visible spectroscopy.
MATERIALS AND REAGENTS:
● Pharmaceutical grade of Paracetamol.
● Acetonitrile.
● Commercial tablets containing 325 mg Paracetamol per tablet.
● UV visible spectroscopy.
● Volumetric flask.
● Pipette.

PRINCIPLE:
Estimation of Paracetamol can be carried out by simultaneous estimation method.
When the sample contains two absorbing species (X and Y ) each of which absorbs
at the Amax of each other. It is possible to determine both the drugs by simultaneous
estimation.

simultaneous equation formula:

X% W/V = 100 { b₁s2-b2s1/b₁s2-b2s1}

Y% W/V = 100 {a1s2-a2s1/a₁b₂-a2b1}
Where:
a Absorbance of component a at A
a2= absorbance of component a at Az
b= Absorbance of component b at A
b2= absorbance of compound b at Az
Si-absorbance of the mixture at A
S2 Absorbance of the mixture at A2
PROCEDURE:
1. PREPARATION OF STANDARD PARACETAMOL STOCK
SOLUTION:
● Take 10mg of Paracetamol and dissolve in 10 ml of acetonitrile.
Concentrations of the solution is 4mg/ml respectively.
● From the above solutions take 1ml and add into 10 ml acetonitrile to get
1mg/ml of the Paracetamol solutions respectively.
● Measure the absorbance of Paracetamol solutions by UV- spectroscopy at
243 nm respectively.

8
2. PREPARATION OF SAMPLE SOLUTION:
● Weigh and powder 5 tablets and take 150 mg equivalent (0.18g) of
Paracetamol
● Take 0.18 grams of the tablet powder into the 50ml volumetric flask. Dissolve
the tablet powder with acetonitrile and filter it and then make up the volume to
50 ml with acetonitrile.
● Take 10ml of the solution into a 100 ml volumetric flask and dilute it to 100ml
with acetonitrile.
● From this take 10 ml into 50 ml volumetric flask and make up the solution to
50 ml with acetonitrile.
Measure the absorbance of 1 mg/ml solution at 243 nm.

9
 MODULE - 2:
SOXHLET EXTRACTION:
INTRODUCTION:
Soxhlet extraction is a modern extraction technique in which we circulate the same
solvent through the extractor several times. It is a type of continuous extraction
technique but we can call it a series of short maceration. Soxhlet extractor needs the
desired compound to be soluble in the solvent at a high temperature. One cycle of
the soxhlet extraction method involves extraction following the evaporation of the
solvent. And theoretically, we can perform this cycle as many times as we want to
get the maximum yield of the desired compound.
Soxhlet extractor makes the extraction process much more efficient than that of the
traditional method.

PRINCIPLE OF SOXHLET EXTRACTION:

Soxhlet extractor extracts the components using the condensed vapours of the
solvent. The condensed vapours come in contact with the sample powder and the
soluble part in the powder gets mixed with the solvent.

SOXHLET EXTRACTION ASSEMBLY:

• As shown in the figure, it has a hot plate of iron, for example, to heat the round
bottom flask containing solvent.

10
• We place RBF (round-bottom flask) on the top of that hot plate. The distillation tube
connects RBF and condenser.

• For better understanding, before looking at the middle part, see the upper part first.
It has a condensation assembly. It has a way to allow cool water in and out.

• Now, the middle part "Thimble" connects to the condensation assembly.

• One more arm connects the thimble and RBF. We call this arm Siphon Tube.

This is how the basic Soxhlet extractor looks like. As a matter of fact, to maximise its
efficiency, we modify this basic assembly.

WORKING:
• First we turn on the heat and the metal plate gets heated.

• The RBF which contains our solvent starts boiling.

• The vapours from the RBF travel from RBF to the condenser via the distillation
tube.

• The condenser condenses the vapours of solvent and those condensed vapours
fall down to thimble.

• We put our sample powder inside the thimble. The powder has to be covered from
the bottom with a cotton ball to avoid powder directly falling into the thimble. And
also cover the powder from the top.

• So, when the condensed vapours fall into the thimble, the powder gets wet with the
solvent and the components which are soluble in the solvent get along with it.

• Siphon connects the thimble to RBF as we saw earlier. The solvent mixture starts
filling thimble and siphon. A point reaches where the siphon starts overflowing under
the influence of gravity.

• Since the Siphon directly connects RBF, the overflowed liquid falls back to RBF.
This marks the first cycle.

• As I mentioned earlier, we can perform as many cycles as we want.

• One thing to mention is, we don't change the solvent for every cycle. And despite
that, when the solvent vaporizes, the components from the sample do not get
vaporised. So, each time we get 100% pure solvent vapours.

11
• When we think that we have exhausted the sample sufficiently, we stop the cycles.
Now we are left with the mixture of solvent and the components from the sample
which are soluble in the solvent.
• Now, we can separate them by further procedures.

SOXHLET EXTRACTION OF VINCA ROSEA:

Synonym: Vinca rosea, Lochnera rosea, Acokanthera
Common Name: Madagascar, periwinkle, old maid, West Indian periwinkle.

Biological Source: It consists of the dried entire plant of Catharanthus roseus linn.

Family: Apocynaceae.

Geographical source: India, Australia, South Africa, North and South America.

chemical constituents: Vincristine, Vinblastine, Indole alkaloids,

Indoline alkaloids,Vindoline, catharanthine-Indole Monomeric alkaloids,
Monoterpenes, sesquiterpene, Indole, Indoline,glycoside.

Action: Cell cycle specific M phase action binds to proteins of mitotic spindle
causing metaphase arrest.
Specific action: Inhibits angiogenesis.
Uses:
1) To treat diabetes.
2) To treat thigh blood pressure.
3) used as disinfectant.
4)Anticancer property.

12
EXTRACTION PROCEDURE:

● Approximately a weighed quantity of dried leaves of vinca rosea (10 gm)

were weighed and placed in a thimble which is present in the extractor.
● The extract is connected to a round bottom flask in the bottom and connected
to a condenser at the top. Heat is supplied by a heating mantle in which the
round bottom flask is placed.
● The round bottom flask consists of porcelain pieces to prevent solution from
bumping due to uneven heating. The temperature is adjusted in such a way
that its equal to boiling point of ethanol that is 78°C.
● Condenser, it consists of an inlet and outlet pipe which is connected to the
water supply.
● 300 ml of ethanol was added as solvent and it's then refluxed for 6 hours in
the heating mantle.
● The extract is then cooled and transferred into a glass bottle covered with
aluminium foil.
● Then it's stored in the refrigerator.

CHEMICAL TEST FOR ALKALOIDS IN VINCA ROSEA EXTRACTION:

TEST OBSERVATION INFERENCE

MAYER’S TEST:
To a small amount of Gives cream colour (or) presence of alkaloids.
crude drug add mayer's precipitate.
reagent (potassium
mercuric iodide solution).

Dragendroff's test:
To the small amount of Gives reddish brown Presence of alkaloids.
crude drug, add colour or precipitate.
dragendorff's reagent
(potassium bismuth iodide
solution).

Wagner's test:
To a small amount of Gives brown or reddish Presence of alkaloids.
crude drug, add Wagner's brown colour or
reagent (iodine-potassium precipitate.
iodide solution).

Hager's test:
To a small amount of Gives yellow precipitate. Presence of alkaloids.
crude drug Hager's
reagent (saturated
solution of picric acid).

13
 MODULE - 3:
FLAME PHOTOMETRY;
INTRODUCTION:

● Several elements like Na, K, Ca, & Ba when burned in the flame emit a
characteristic flame. The colour is a characteristic of a particular element. It
means, metal/metallic salts excited in a flame in flame photometry.
● Specific λ which is, Characteristics of elements of the visible and UV radiation
untitled out, are observed in qualitative analysis.

ELEMENT EMISSION FLAME COLOUR

Sodium 589 Yellow colour

Potassium 766 Violet colour

Barium 554 Light green colour

Calcium 622 Orange colour

Lithium 670 Red colour

● calcium measured by using the Ca(OH)2 bond emission at 623 nm as Ca

main atomic emission occurs at 423 nm.

14
CONCEPT OF FLAME PHOTOMETRY:

● Introduction of metallic salt solution.

● Production of liquid droplet - Nebuliser.

● Evaporation of liquid (formation of solid residue) - Dissolution by flame.

● Gaseous atoms - Vaporisation by flame (thermal energy).

● Dissociation of free neutral atoms - Atomization by flame.

● Excitation of atoms - Excitation by heat of the frame.

● Emission of radiation from the excited atoms.

● Emission Measurement & intensity of emitted radiation.

PRINCIPLE INVOLVED:

Principle involved in flame photometry is based on measurement of intensity of light

emitted when a metal is introduced into the flame.

The (A)of colour tells us what the element is, and colour intensity tells us about how
much of element present in that.

INSTRUMENTATION:
1) Burners
2) Mirrors
3) Slits
4) Monochromators
5) Filters
6) Read out device.

Burners:
a) Mecker burners:
● Produces relatively low temperature and low excitation energies, generally
used for study of alkali metals only.
b) Total consumption burner:

15
 ● In this the fuel and oxidant are hydrogen and oxygen gases respectively.
Because all the sample that enters the capillary tube will enter the flame
regardless of droplet size, as the name suggests

C) premix of Laminar flow burns:

● The flame produced by us non-turbulent, noiseless and stable. Easy
decomposition of sample results in efficient Atomization of sample in flame
d)Lundegårdh burners:
● Sample used must be in liquid form
● It is aspirated into the spray chamber. Large droplets condense on the side
and drain away, small droplets and vapourised samples are swept into base
of flame in the form of cloud impact bond, ultrasonic vibrations thermo spray
heaters devices used for flame entrance in the nebulization stage.
e) shielded burners:
● In which the flame was shielded from the ambient atmosphere by steam of
inert gas.
f)Nitrous oxide acetylene flames:
● This flame was superior to the other flames for efficiently producing tree
atoms.
g)Sequence of events in a flame :
[Link] (or) other solvent is evaporated, leaving minute particles of dry salt.
2. The dry salt is vapourised /converted into gaseous salts.
3. A particle of gaseous molecules is progressively dissociated to get free neutral
atoms /radicals.
4. A part of neutral atoms may be thermally excreted/even ionised.
5. A portion of neutral atoms /radicals in flame may combine to form new gaseous
compounds.
[Link]:
● In order to maximise the amount of radiation used in the analysis, a mirror is
located behind the burner to reflect the radiation back to the entrance slit of
the monochromator.
● The reflecting surface mirror is front faced.
● Easily scratched and subject to chemical attack great care must be taken.
3) Slits:
● Entrance and exit slits are used by before and after the dispersion elements.
● The entrance slits cut out most of the radiation from the surroundings and
allows only the radiation from the flame and mirrored reflection to enter the
optical stream.
● The exit slit is placed after the monochromator & allows only a stretched
lambda range to pass through the detector.
4) Monochromator:
● In a simple way, a monochromator is a prism.
● In the expensive grating monochromator is used.
● It works as a converter of a series of parallel lines cut into a plane surface.

16
5) Filters:
● Filters used in the place of slit and monochromator kept b/w flame and
detector, the radiation of desired lambda from the flame will be entering
will be entering the detectors and measured. The remaining undesired (A) will be
absorbed by filter and not measured.
6)Detectors:
● The detector shield is sensitive to radiation if all lambda fall on the detector.
● Photomultiplier detectors are employed.
CALIBRATION OF FLAME PHOTOMETER USING SODIUM AS
STANDARD:
PREPARATION OF SODIUM CHLORIDE STOCK SOLUTION:
Weigh accurately 0.127gm of analytical reagent grade, dry, sodium chloride salt.
● Place 1-2gm salt on watch glass, heat at 110c for 1 hour.
● After completion of drying place accurately weighed quantity of salt and
makeup with distilled water up to 50 ml in a standard volumetric flask.
● Results in formation of 1000 PPM sodium solution.
● Take 5ml of above prepared 1000 PPM sodium and make up with distilled
water up to 50 ml in a standard volumetric flask.
● Results in formation of 100 PPM sodium solution.
● Take 25 ml of above prepared 100 PPM sodium and make up with distilled
water up to 250 ml in a standard volumetric flask.
● Results in formation of 10 PPM sodium solution.
● Prepare 2, 4, 6 & 8 PPM sodium solution in the same manner.
● The detector shield is sensitive to radiation if all lambda falls on the detector.
● Photomultiplier detectors are employed.

17
 MODULE - 4:
WATER FOR INJECTION:
INTRODUCTION:
● Water for Injection, commonly referred to as WFI.
● It is a solvent that is used to dilute other medications or solutions that will be
injected into the body.
● By regulation, it can contain very little biological or chemical agents that
inhibit bacteria or microorganism growth.
● Water for Injection (WFI) is utilised as a pharmaceutical vehicle to deliver
drugs intravenously (parenteral preparations), but also as a fluid replenisher
after an appropriate solute.
● Additionally, WFI water can be used for inhalation medications (breathed
directly into the lungs) and some ophthalmic (eye health) products.

DEFINITIONS OF DIFFERENT TYPES OF WATER:

Potable water:

● Potable water is water that is safe to drink. and can be used for food
preparation. It is filtered and treated properly and is free from all
contaminants.
● Potable water meets Local established drinking water Standards it should be
colourless and Odourless.
● It should be Transparent.
● free from impurities such as suspended solids.

Distilled water:

● Distilled water is a water that has been purified using a process called
distillation.
● In this process, water is boiled into vapour and then condensed back into
liquid in a separate container.
● The impurities in the water. that do not boil below (or) at the boiling point of
water remain in the original container.

Double distilled water:

● Double distilled water is water that has been distilled twice.

● It's produced by boiling water and then condensed the vapour into a clean
container and the process is repeated.

18
 ● This process removes more than 99.9% of contaminants including - inorganic
salts,organic matter, microorganisms, soluble gases,heavy metals.
Double distilled water is used in laboratories when single distillation is not pure
enough for research applications.

Pyrogen free water:

Pyrogen free water is water that is free of fever -producing proteins, these proteins
are released from bacteria, virures,(or) destroyed cells of the body. They can raise
the boy's temperature, which is a significant indicator of disease.
● Pyrogen free water is also known as Endotoxin free water.

PREPARATION OF WATER FOR INJECTION:

ALL GLASS PYROGEN FREE WATER DISTILLATION STILL:

All glass pyrogen free water distillation units produce highly treated and disinfected
water for laboratory usage. The distillation process removes minerals and
microbiological contaminants and can reduce levels of chemical contaminants.

WORKING PRINCIPLE:

Evaporation and condensation are the main mechanisms that drive distillers. Water
is transformed into vapour by adding thermal [Link] the collected water vapour
is then sent via a condenser, which is cooled and transformed into liquid. The
condensed water is then collected and placed in an additional storage tank.

CONSTRUCTION DETAILS:

● The distillation apparatus consists of a flask with heating elements embedded

in glass and fused in a spiral type coil internally of the bottom and tapered
round glass.
● joints at the top double walled condenser with ground glass joints, suitable to
work on 220 volts, 50 cycles AC supply.

power requirements: To work on 220/230 Volts, 50 Hz single phase AC supply.

flask capacity: 3 litres,5 litres,10 litres

optional accessories:Automatic Low Water Level Cut-off Device, in case water
supply is stopped Special grade rubber tubing (coil of 10 metres).

19
WORKING PROCEDURE:

● Potable (drinking water) water is manually or automatically fed into the distiller
unit's boiling chamber.
● A heating element in the boiling chamber heats the water until it boils.
● The steam rises from the boiling chamber. Volatile contaminants (gases) are
discharged through a built-in vent Minerals and salts are retained in the
boiling chamber as hard deposits or scale.
● The steam enters a coiled tube (condenser), which is cooled by cool water.
● Water droplets form as condensation occurs.
● The distilled water is collected in a storage tank. If the unit is an automatic
model, it is set to operate to fill the storage tank.
STERILITY TEST FOR WATER FOR INJECTION:

PREPARATION OF CULTURE MEDIA:

SOYABEAN CASEIN DIGEST MEDIUM:

COMPOSITION:
Pancreatic digest of casein. 17.0g.
Papaic digest of soyabean meal. 3.0g.
Sodium chloride. 5.0g.
Dipotassium hydrogen phosphate (K₂HPO4). 2.5g.
Dextrose monohydrate/anhydrous. 2.5g/2.3g.
Distilled water too. 1000ml.
pH of the medium after sterilisation. 7.3±0.2.

20
PROCEDORE:
● Dissolve the solids in distilled water warming slightly to effect [Link] to
room temperature and add if necessary sufficient 1m sodium hydroxide
solution so that after sterilisation the medium will have a pH of 7.3 +/- 0.1.
filter the solution if necessary and distribute into suitable container and
sterilise in autoclave at 121°c and at 15 pounds of pressure for 20 mins.
● Use soyabean-casein digest medium by incubating it at 30 to 35°c for
18 to 24 hrs.

STERILISATION OF CULTURE MEDIA BY MOIST HEAT

STERILISATION (AUTOCLAVE):

PRINCIPLE:
An autoclave is a device that works on the principle of moist heat sterilisation. where
in, saturated steam is generated under pressure in order to kill microorganisms such
as Viruses, and even heat resistant endospores from various types of
[Link] is done by heating the instruments within the device to
temperatures suppressing the boiling of water.

This process is also embodied by gas laws, which basically states that the higher the
pressure is within the device,the higher temperature increases in the other words,
pressure and temperature are directly proportional to each other.

Steam sterilisation is effective because the moisture of the steam helps coagulate
the proteins that microbes thrive on coagulating the proteins. disables the microbes
and eventually kills them.

Autoclaves typically yield temperatures of about 121°[Link] at 15 pounds of

[Link] about 15 to 20 mins to complete the sterilisation process.

PROCEDURE:
● The materials to be sterilised should be kept in a material which will not create
any obstacle to steam penetration and removal of [Link] can be
packed in tubes, bottles closed with cotton stoppers.
● place sufficient water up to the required level. in the chamber and place the
articles on the perforated tray just above it.
● close the door to seal it completely. Open the manual discharge tap and turn
on the source of heat.
● Allow the steam and air to escape freely. through the discharge tap for 5
mins.
● And close the discharge tap and wait till the pressure is reached to the
required level.
● Total sterilising period of 15-20 mins at 15 pounds of pressure.

21
PRECAUTIONS:
● Bottles used for packing must be strong and volumes should not be more than
500ml for sealed bottles.
● large bottles to be closed with loosely applied caps(or)Cotton wool stoppers.
● Bottles should be kept in a regular way and should not be over crowded.
● Do not open the discharge tap [Link] the steam [Link]
open it before the pressure reaches to atmospheric pressure and at the same
time do not deliver the opening once it is below atmospheric pressure.
● Maintain the water level above the heating element before using.

ASEPTIC TRANSFER OF TEST SAMPLE INTO THE CULTURE

MEDIA BY USING THE LAMINAR AIR FLOW:

Laminar air flow:

The air flow which is linear and positive up to working surfaces and thus prevents
contamination of surrounding viable and non viable. particulate matter in aseptic
handling.

PROCEDURE:
● Tum on the switch of uv light leave the uv for at least 30 [Link] then turn off
the UV light.
● Turn on the switch of visible light and air flow.
● wipe the working surface as well as the glassware and equipment placed
inside the cabinets with 70% ethanol.
● light the spirit lamp (or) Bunsen burner inside the cabinet.
● And heat the inoculating loop by using a spirit lamp and also heat the mouth
of the sample test tube.

22
● Take a loop full of sample by inserting the inoculating loop into the test sample
solution.
● And transfer it into culture media by placing the inoculating loop
perpendicularly.
● And incubate the test tubes in the incubator at 30°C-35°C for 18 to 24 hours.

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 MODULE - 5:
PREPARATION OF TABLETS:
INTRODUCTION:
Tablets are the solid pharmaceutical dosage form containing drug/s and
excipients. They are manufactured primarily by compression method, with a limited
number prepared by moulding methods. Tablets can be administered through oral,
sublingual, buccal and vaginal route. Tablets may vary in size, shape, weight,
thickness, hardness etc based on their use and on the amount of active ingredient.
They are cheaper among all other oral dosage forms. Tablets have the highest dose
precision and least content variability among all the oral dosage forms.
GRANULATION METHODS:
● In the pharmaceutical industry, granulation is the process in which powder
particles adhere to form large entities called granules. Granules are prepared
by collecting particles together and developing bonds between them. The
bonds are formed either by compression or by using binding agents.
● Granulation is used in manufacturing of tablets and pellets. The advantage of
granules is that they flow evenly through the hopper of the tablet machine. On
compression, granules form a sound tablet. Granules are also uniform in
composition.
● Granules are prepared by three methods.
a. Wet granulation method.
b. Dry granulation method.
c. Direct compression.
WET GRANULATION METHOD:
Wet granulation method of tablet production involves the following processing steps:
[Link] and mixing of formulation ingredients with excipients (excluding
the lubricant):
● In this step weighing, sifting and drug substance(s) and excipients (such as
bulking [Link] or diluent, and disintegrant are mixed into a powder mixer.
These ingredients are mixed using a mixer to get a uniform powder mix.
2. Preparing the damp mass:
● This step involves addition of the binder solution with the powder mixture to
form a coherent mass. Insufficient binder cause poor adhesion, capping and
yielding soft tablets while excessive binder solution yields hard tablets with
slow disintegrating properties.
3. Wet screening:
● The wet massed powder blend is screened using sieve no 8 or 10. This may
be done by hand or with suitable equipment. The wet granules formed are
spread evenly on trays and dried in an oven.
4. Drying of moist granules:
The wet granules are dried in an hot air oven at 60°C. The drying temperature and

24
The duration of the drying process is also adjusted.
5. Sizing the granulation by dry screening:
The dried granules are passed through a sieve no 20 to get uniform size granules.
6. Lubrication of granules:
● Appropriate quantity of lubricant is mixed with the granules. The remaining
amount of disintegrants are also added at this stage.
7. Compression of granules into tablets:
● The mixed granules are compressed in a single punch or multi-station tablet
press fitted with the appropriate punches and dies.
DRY GRANULATION METHOD:
It is also known as the Slugging and Double Compression method. The dry
granulation process is used to form granules without using a liquid solution because
the product to be granulated may be sensitive to moisture and heat. Dry granulation
method of tablet production involves the following processing steps:
1. Weighing formulation ingredients:
● The appropriate quantities of formulation ingredients and excipients are
weighed on analytical balance.
2. Mixing of formulation ingredients:
● The dispensed formulation ingredients are mixed in a powder mixer until a
uniform powder mix is achieved. The half quantity of lubricant is added at this
stage to enhance powder flow during slugging and to prevent sticking of
compressed powder on the die.
3. Compression of mixed powders into slugs:
● Here, the mixed ingredients are compressed into flat large tablets. This step is
called precompression or slugging. The pressure used to produce the slugs is
usually less than that used in the final compression. A large tablet which is
also known as a 'slug' is produced in a heavy-duty tableting press or the
powder is squeezed between two rollers to produce a sheet of material by
using a roller compactor..
4. Milling and sieving of slugs:
● This step involves breaking of slugs into smaller pieces using a hammer mill
or other conventional milling equipment. The milled slugs are screened to
produce uniform granules.
5. Mixing with disintegrant and lubricant:
● After screening, the remaining lubricant and other extragranular excipients
such as disintegrant, glidant etc., are incorporated to the granulation and
mixed gently to achieve a uniform blend.
6. Compression of granules into tablet:
The mixed granules are compressed into a tablet using a tablet machine.
DIRECT COMPRESSION:
As the name indicates, the processes involved in the manufacture of tablets by direct
compression. This method involves following steps.

25
1. Weighing and Premilling of formulation ingredients (active drug substance and
excipients).
2. Mixing of active drug substance with the powdered excipients (including the
lubricant).
3. Compression of the mixed powders in a rotary press to tablets.

TABLET COMPRESSION MACHINES:

● The dried granules are compressed into tablets in a machine known as Table
compression machine or Press.
● There are different types of presses used for this purpose.
1. Single Punch Tablet machine.
2. Multi Station rotary press.
SINGLE PUNCH TABLET MACHINE:
● It is also called eccentric press or single station press. Single punch tablet
machines include a die and a pair of upper and lower punches. The functional
part of Single Punch Tablet machine are
a. Hopper: It is used to supply granules/ powder mixtures to die prior to
compression. The hopper can be filled manually or by using mechanical equipment.
[Link]: It permit lower punch to come close to upper punch so that compression of
granules take place.
c. Die cavity:The die cavity is where the powder granules are compressed into
tablets.
d. Punches: This includes upper punch and lower punch. The amount of powder
filled into the die is adjusted by the position of the lower punch. Punches are used to
compress the powder into tablets of various shapes within the die.
e. Cam track: This helps to guide the position/movement of the punches.
f. Capacity regulator: This is used to adjust the lower punch position to allow only
the required quantity of granules by the die.
g. Ejection regulator:This facilitates the ejection of the tablet from the die cavity
after compression.

26
WORKING:
● The upper punch moves up and lower punch moves down to create a cavity in
the die. The feed from the hopper falls into the die cavity. The upper punch
moves down to compress the granules/powder mixture into tablets. The upper
punch moves up and the lower punch also moves upwards to eject the
compressed tablet. The whole process repeats again and again until the feed
material is exhausted.
● The machine is easy to operate. The output is about 200 tablets per minute.
Single punch tablet machines are ideal for small batch production. Single
punch tablet press utilises a high amount of pressure to reduce weight
variations between tablets.
MULTI STATION ROTARY PRESS:
Multi-station press is termed as rotary because it comprises approximately 60 sets of
dies and punches which rotate to compress granules/powder mixture into tablets of
uniform size, shape and uniform weight. This is used for large scale production. The
machine increases the output of tablets. The machine produces 1200 tablets in one
minute.
● Parts of Multi Station Rotary press:
Upper and Lower Turrets: It is that portion of the head that holds upper and lower
punches.
Lower cam track: It is used to guide the lower punch to ensure the die cavity is
overfilled.
Feeder or Feed Frame: It is used to feed the powder or granules into the die.
Cam tracks: It guides the movement of both the upper and lower punches.
Ejection cam: It is used to facilitate ejection of tablets from the die cavity by moving
lower punch upward.
Take-off blades: It helps to push the tablets to a discharge chute, which is collected
in a container.

27
WORKING:
● As the name indicates "rotary tablet press" it has a rotating turret. The powder
or granules are placed in the hopper. From where it will flow to the tooling
system of the machine ready for compression. The material is then fed into
several dies [Link] is accurate filling of the die cavities. The
machine removes excess powder to avoid any form of inconsistencies.
Therefore, desired volume and weight of powder is compressed into tablets.
The upper and lower punches exert a predetermined amount of pressure that
compresses tablets to the right size and depth.
● As the head revolves, the dies come under the feed frame and get filled.
These granules get compressed when upper and lower punches come close
together. The pre-compression process removes any traces of air that might
be within the powder particles. While the main compressor exerts an
appropriate amount of force that compacts powder to a desired thickness and
hardness. The pressure is exerted on the punches by a series of pressure
rolls.
● It is a continuous process. The upper cams pull the upper punches back to
their initial position and the lower punches are also lifted up to eject processed
tablets out of the die cavity. The scraper will then remove the tablet from the
compression machine to the discharge chute. It is considered the end of one
complete cycle of the tablet compression machine.

QUALITY CONTROL TEST FOR TABLETS:

Official Tests:
● Weight variation
● Disintegration test
● content uniformity
Non-Official Tests:
● Hardness
● friability
OFFICIAL TESTS:
WEIGHT VARIATION TEST;
To calculate uniformity of weight, 20 tablets are randomly selected and weighed. The
average weight is determined and the individual weight of each tablet is also
calculated. Then the individual weight of each tablet is compared with average
weight. Not more than two of individual weight may deviate from average weight by
more than percentage deviation as given in below table and none should deviate
more than twice that percentage.
Average weight of tablet deviation Percentage

80 mg or less 10

More than 80 mg and less than 250 mg 7.5

28
 250 mg or more 5

Disintegration test for tablet:

Disintegration testing determines the time required for the breaking of tablets when
placed in a liquid medium. The apparatus used to perform the test is known as
disintegration test apparatus. The apparatus consists of a water bath which is filled
with water up to the mark mentioned. Place 1000 ml beakers into a water bath. A
basket rack holding six plastic tubes open at the top. The bottom is covered with a
10 mesh screen. The basket rack assembly is suspended in liquid medium in 1000
ml beakers. The temperature of liquid is maintained at 37°C. One tablet is placed
into each tube. The assembly moves up and down at a specified rate (30 times per
minute). A cylindrical disk made of transparent plastic is also placed over the tablet.
The disc should impart little pressure on the tablet. The time to disintegrate the tablet
and fall through the screen is noted.

The beaker is filled in such a way that the highest point of wire mesh remains about
2.5 cm below the surface of liquid while its lowest point remains about 2.5 cm above
the bottom of the beaker.
The rate of disintegration varies from tablet to tablet
Content Uniformity:
According to USP, 10 tablets are assayed individually. Out of this 9 should contain
not less than 85% or more than 115% of the labelled drug content. The 10th tablet
should contain not less than 75% or more than 125% of the labelled content. If this
condition is not fulfilled then other tablets are assayed individually and not of any
tablet may fall outside the range of 85-115%.
NON-OFFICIAL TESTS:
Hardness:
The tablet to be tested is placed between jaws as seen in figure. The reading on the
pressure dial will be zero. Then press the pliers-like handle with your hands. The
reading on the pressure dial is noted which indicates the force required in kilograms
or pounds to break the tablets.

29
Friability Test:
This test is applicable for compressed uncoated tablets. This test is done to
determine physical strength of tablets especially during handling and transportation
The instrument used to perform this test is known as "Roche's Friabilator".

The instrument consists of a drum or plastic chamber having diameter 283-291 mm

and depth is 36-40 mm. A central ring is present whose outer diameter is 24.5 to
25.5 mm. The rotation speed of the drum is 25 rpm. Carefully dedust the 20 tablets
and weighed them Placed them in a plastic chamber and rotated it for 4 minutes or
set 100 number of counts During each revolution tablets fell from a distance of 6
inches. After 4 minutes or 100 counts removed tablets from the chamber and again
weighed the tablets. Loss in weight indicates friability. It is expressed in percentage.
A good quality tablet will show loss in weight is less than 0.8%,

30
 MODULE - 6:
SOXHLET EXTRACTION OF CHRYSANTHEMUM
MORIFOLIUM:
INTRODUCTION TO SOXHLET EXTRACTION:
Soxhlet Extraction:
● A soxhlet extraction is a continuous process of extraction with a hot organic
solvent.
● A soxhlet extractor is a piece of laboratory apparatus invented in 1879 by
Franz von soxhlet it was originally designed for the extraction of lipid from a
solid material, typically soxhlet extraction is used when the desired compound
has a limited solubility in a solvent and the impurities is insoluble in that
solvent.
● It allows for unmonitored and unmanaged operation while efficiently recycling
a small amount to dissolve a larger amount of material.
DESCRIPTION:
A soxhlet extractor has three main sections:
● A percolator (boiler and reflux) which circulates the solvent.
● A thimble (usually made up of thick thick filter paper) which retains the solids
to be extracted.
● And a syphon mechanism which periodically empties the thimble.

31
ASSEMBLY:
● Thimble: The source material containing the compound to be extracted is
placed inside the thimble. The thimble is loaded into the main chamber of the
Soxhlet extractor.
● Distillation flask or Round Bottom Flask (RBF): The extraction solvent to
be used is placed in a distillation flask.
● Heating mantle: The flask is placed on the heating element.
● Reflux condenser: A reflux is placed atop the extractor.
● Water inlet & outlet: Attached in the inlet & outlet opening of condenser.
WORKING PROCEDURE:
● Solid material placed in thimble.
● Soxhlet extractor is placed onto a flask containing the extraction solvent
Soxhlet equipped with a condenser.
● The solvent is heated to reflux.
● The solvent vapour travels up a distillation arm and floods into the chamber
housing the thimble of solid.
● Solid material in the chamber slowly fills warm solvent.
● Desired compound dissolves in the warm solvent.
● When the Soxhlet chamber is almost full, the chamber is emptied by the
syphon. The solvent runs back down to the distillation flask.
● This cycle may be allowed to repeat many times, over hours or days.
● During each cycle, a portion of the compound dissolves in the solvent.
● After many cycles (72 hours) the desired compound is concentrated in the
distillation flask.
● After extraction the solvent is removed, typically by means of a rotary
evaporator, yielding the extracted compound.
● The non-soluble portion of the extracted solid remains in the thimble, and is
usually discarded.

EXTRACTION PROCEDURE FOR CHRYSANTHEMUM

MORIFOLIUM:
INTRODUCTION:
Synonym: crown daisy, corn marigold.
Common name: Mums (or) chrysanths.
Family: Asteraceae
Geographical Source: Native to the northern hemisphere, chiefly Europe & Asia.
Chemical constituents: The two main constituents are terpenoids & flavonoids.
The flavonoids present are apigenin,luteolin, quercetin and their derivatives such as
flavonols & flavonol glycosides.
Main action: It has antioxidant and anti-inflammatory effects.

32
Uses: It is used to treat chest pain (angenia),it also treats blood pressure, fever,
cold, headache.
It is commonly used as an herbal treatment for hypertension & inflammation.
It is also useful in treating type 2 diabetes.

PROCEDURE:
● Approximately a weighed quantity of dried petals of chrysanthemum
morifolium are dried and powdered.
● 50 grams of powdered Sample is placed in the thimble which is present in
Extractor.
● The Extractor is connected to a round bottom flask in the bottom that contains
porcelain pieces in it to prevent Solution from bumping.
● At the top a condenser is connected.
● Heat is supplied by the heating mantle in which RBF is placed.
● The condenser has an inlet and outlet which is connected to water supply.
● The solvent contains 90 ml of water & 90 ml of Alcohol & is then refluxed for
5-6 hours in the heating mantle.
● The Extract is then cooled and transferred to a glass container & then stored
in the refrigerator.
EXTRACTION PROCEDURE FOR FRESH TURMERIC
RHIZOME:

INTRODUCTION:
Synonym: curcumin, curcuma, curcuma aromatica.
Common name: Haldi, Manjal.
Family:zingiberaceae.
Geographical Source: Native to Southern India and [Link] cultivated on
mainland in the islands of indian Oceans.
Chemical constituents: turmeric contains curcumin along with other constituents
known as "Curcuminoids".
● demethoxy curcumin, cyclocurcumin.

33
 ● Curcumin has a brilliant yellow colour at pH 2.5 & takes red colour at pH>7.

USES:
● It is used as anti-inflammatory and to treat digestive and liver problems, skin
diseases, and wounds.
● It is also used as colouring and flavouring agents in cooking.

PROCEDURE:

● 30 grams of partially dried & weighed turmeric rhizomes are taken & packed
in the thimble.
● Then the thimble containing the Sample is placed in the extractor and .
connected to the RBF, at the bottom.
● Round bottom flask contains porcelain pieces which prevents solution from
bumping.
● At the top of the RBF a condenser is connected which has an inlet & outlet
for water supply.
● A mixture of 100ml Alcohol & 50ml water was taken as the solvent for
Extraction.
● The whole apparatus was placed in the heating mantle and the temperature
was set up to 60°C and then refluxed it for 5-6 hours.
● After the completion of extraction, the extract was collected, cooled and
transferred to a glass container & Stored in refrigerator.

34