3. Materials and Methods

CHAPTER THREE
MARETIALS AND METHOD
3. Materials and Methods

3.1. Materials: Albendazole, Mebendazole, Sodium starch glycolate, lactose monohydrate, starch,
Mannitol, Sodium saccharine, Magnesium stearate, Talc, Aerosil.
3.1.1. Albendazole: Albendazole, also known as albendazolum, is a medication used for the treatment of
a variety of worm infestations. is an orally administered anthelmintic drug. Chemically, it is methyl 5-
(propylthio)-2-benzimidazolecarbamate. Its molecular formula is C12H15N3O2S. Its molecular weight is
265.34 [49]. Albendazole is used for giardiasis, trichuriasis, filariasis, neurocysticercosis, hydatid
disease, pinworm disease and ascariasis.
3.1.2. Mebendazole: Mebendazole is methyl 5-benzoylbenzimidazole-2-carbamate,molecular formula of

C16H13N3O3. Mebendazoleis a white to slightly yellow powder with a molecular weight of 295.29 [50].
3.1.3. Sodium Starch Glycolate: Sodium starch glycolate is the sodium salt of carboxymethyl

ether. Sodium starch glycolate absorbs water rapidly, resulting in swelling which leads to rapid
disintegration of tablets and granules [51]. It is used as a disintegrant, a suspending agent and as a gelling
agent.
Sodium starch glycolate is used as a pharmaceutical grade dissolution excipient for tablets and capsules.
Sodium starch glycolate absorbs water rapidly, resulting in swelling which leads to rapid disintegration of
tablets and granules. It is used as a disintegrant, a suspending agent and as a gelling agent. Without a
disintegrant, tablets may not dissolve appropriately and may effect the amount of active ingredient
absorbed, thereby decreasing effectiveness.
3.1.4. Lactose Monohydrate: Lactose (C12H22O11) is milk sugar. It is a disaccharide composed of one
galactose and one glucose molecule. In the pharmaceutical industry, lactose is used to help form tablets
because it has excellent compressibility properties. It is also used to form a diluent powder for dry-
powder inhalations. Lactose may be listed as lactose hydrous, lactose anhydrous, lactose monohydrate, or
lactose spray-dried.
Lactose is a naturally occurring simple carbohydrate, or sugar, found only in the milk of mammals. For
this reason, it is also commonly referred to as “milk sugar.” All commercial lactose is obtained from the
milk of cows as a by-product of the dairy industry. Chemically, lactose is the disaccharide of the simple
sugars D-galactose and D-glucose. In other words, the lactose molecule comprises one molecule of D-
galactose chemically linked to one molecule of D-glucose. Lactose exists in two isomeric forms, known
as alpha and beta (designated a-lactose and b-lactose).
3.1.5. Starch: Starch is nontoxic and nonirritant properties, as well as low cost, ease of modification, and
versatility in use have placed starch in a leading position among polymers used as
apharmaceutical excipient. In many conventional tablets and capsules, starch is used as a diluent,
disintegrant, binder, and lubricant.
3.1.6. Mannitol: Mannitol (C6H8(OH)6) is used in pharmaceutical products as a sweeting agent, tablet

and capsule diluent, excipient for chewable tablets, a tonicity agent, and as a vehicle (bulking agent) for
lyophilized preparations. Mannitol has a cooling effect often used to mask bitter tastes, and may be used
in gums and candies.
Excessive consumption of mannitol may lead to a laxative effect, but the small amount used in
pharmaceutical manufacturing processes would not normally pose this risk. Mannitol is deemed a safe
food ingredient. Mannitol does not lead to elevated levels of blood sugar, as glucose may
3.1.7. Sodium Saccharin: Saccharin, also known as saccharin sodium or benzosulfimide, is 300–500
times sweeter than sucrose (sugar) in dilute aqueous solution. It is used as a noncaloric sweetening agent
(sugar substitute.
3.1.8. Magnessium Stearate: Magnesium stearate (Mg(C18H3502)2 or octadecanoic acid) is a solid,

white powder at room temperature.
It is a FDA-approved inactive ingredient commonly used in the pharmaceutical industry as a diluent for
the manufacture of tablet, capsule, and powder dosage forms.
Magnesium stearate is generally recognized as safe by the FDA. Magnesium stearate exists as a salt form
and is useful for it's lubricating properties for capsules and tablets in industry. It is used to help prevent
pharmaceutical ingredients from adhering to industry equipment. Magnesium stearate may be derived
from both plant and animal sources.
3.1.9. Talc: Talc is a clay mineral composed of hydrated magnesium silicate with the chemical formula

Mg3Si4O10(OH)2. Talc in powdered form, often in combination with corn starch. It demonstrates the high
functionality of multiple excipients because it has been used as filler, lubricant and glidant in the
pharmaceutical formulations.
In the pharmaceutical industry it is used as an anticaking agent to improve powder flow

intablet compression.
3.1.10. Aerosil: Aerosil colloidal silicon dioxide has been used as a glidant to optimize the flow of
powders since the earliest days of direct compression. The small Aerosil fumed silica particles “coat” the
larger excipient and active ingredient particles, thus reducing van der Waals attractive forces between
them. Fumed silica serves as a universal thickening agent and an anticaking agent (free-flow agent)
in powders. Like silica gel, it serves as a desiccant. It is used in cosmetics for its light-diffusing
properties. It is used as a light abrasive.
3.1.11. Coloring Agent: The primary function of colour is that it allows customers to recognize one pill
from another and also between prescription/non-prescription drugs and other capsules or tablets. Elderly
patients often get confused when all pills are of the same size, colour and shape. coloring agents are
mainly used to impart a distinctive appearance to the pharmaceutical dosage forms.
3.1.12. Flavouring Agent: A flavor, as used in the pharmaceutical industry for inactive ingredients, refers
to natural or artificial tastes, which may include fragrances and colors of the flavoring. Flavors are used
for orally consumed products such as syrups, chewable tablets, suspensions, or gums that impart
beneficial therapeutic effect, as well.
The U.S. Code of Federal Regulations describes a natural flavorant as: the essential oil, oleoresin, essence
or extractive, protein hydrolysate, distillate, or any product of roasting, heating or enzymolysis, which
contains the flavoring constituents derived from a spice, fruit or fruit juice, vegetable or vegetable juice,
edible yeast, herb, bark, bud, root, leaf or similar plant material, meat, seafood, poultry, eggs, dairy
products, or fermentation products thereof, whose significant function in food is flavoring rather than
nutritional.
Artificial flavors include any substance, the function of which is to impart flavor, which is not derived
from a spice, fruit or fruit juice, vegetable or vegetable juice, edible yeast, herb, bark, bud, root, leaf or
similar plant material, meat, fish, poultry, eggs, dairy products, or fermentation products thereof
It Improves the desirable characteristics of taste, texture, and overall palatability of formulations with
Spectrum's selection of pharmaceutical flavoring agents.
3.2. Apparatus and Machine:
3.2.1. Beaker: Used in biological experiment.
3.2.2. Conical Flask: Used in dissolution test, biological experiment.
3.2.3. Round Bottom Flask: Used in HPLC analysis, biological experiment.
3.2.4. Glass Rod: Used in biological experiment and others.
3.2.5. Weighing Balance: Used to measure the weight of materials.
3.2.6. pH Meter: Used for preparing buffer.
3.2.7. Funnel: Used in dissolution test, for measuring angle of repose.
3.2.8. Rotary Tablet Punching Machine: Used to prepare tablets from powders by compression.
3.2.9. Measuring Cylinder: Used for measuring bulk density and tapped density.
3.2.10. Hardness Tester Machine: Used to measure the hardness of tablets.
3.2.11. Dissolution Tester Machine: Used to perform the dissolution test of tablets.
3.2.12. Disintegration Tester Machine: Used to perform the disintegration test of tablets.
3.2.13. Friability Tester Machine: Used to perform the friability test of tablets.
3.2.14. FTIR Analysis Machine: Used to perform FTIR analysis of powders.
3.2.15. SEM Analysis Machine: Used to perform SEM analysis of tablet surface.
3.2.16. HPLC Machine: Used for HPLC analysis of powders and tablet.
Figure 3.1: Different Apparatuses

3.3. Methods: Here we use only aqueous granulation method to formulate the combination of
Albendazole and Mebendazole.
3.3.1. In vitro Anthelmintic Assay
Experiment before Formulation
For evaluating the best combination ratio of Albendazole and Mebendazole, we had to do experiments on
Earthworms ( Lumbricus terrestris).
An earthworm is a tube-shaped, segmented worm found in the phylum Annelida. They have a world-wide

distribution and are commonly found living in soil, feeding on live and dead organic matter. An
earthworm's digestive system runs through the length of its body. It conducts respiration through its skin.
It has a double transport system composed of coelomic fluid that moves within the fluid-filled coelom and
a simple, closed blood circulatory system. It has a central and a peripheral nervous system. The central
nervous system consists of two ganglia above the mouth, one on either side, connected to a nerve cord
running back along its length to motor neurons and sensory cells in each segment. Large numbers of
chemoreceptors are concentrated near its mouth. Circumferential and longitudinal muscles on the
periphery of each segment enable the worm to move. Similar sets of muscles line the gut, and their
actions move the digesting food toward the worm's anus.
Earthworms are hermaphrodites: each individual carries both male and female sex organs. As
invertebrates, they lack either an internal skeleton or exoskeleton, but maintain their structure with fluid-
filled coelom chambers that function as a hydrostatic skeleton.
"Earthworm" is the common name for the largest members of Oligochaeta (which is either a class or a
subclass depending on the author). In classical systems,they were placed in the order Opisthopora, on the
basis of the male pores opening posterior to the female pores, though the internal male segments are
anterior to the female. Theoretical cladistic studies have placed them, instead, in the suborder Lumbricina
of the order Haplotaxida, but this may again soon change. Folk names for the earthworm include "dew-
worm", "rainworm", "night crawler", and "angleworm" (due to its use as fishing bait).
Larger terrestrial earthworms are also called megadriles (which translates to "big worms"), as opposed to
the microdriles ("small worms") in the semiaquatic families Tubificidae, Lumbricidae, and Enchytraeidae,
among others. The megadriles are characterized by having a distinct clitellum (which is more extensive
than that of microdriles) and a vascular system with true capillaries.
Depending on the species, an adult earthworm can be from 10 mm (0.39 in) long and 1 mm (0.039 in)
wide to 3 m (9.8 ft) long and over 25 mm (0.98 in) wide, but the typical Lumbricus terrestris grows to
about 360 mm (14 in) long. Probably the longest worm on confirmed records is Amynthas
mekongianus that extends up to 3 m (10 ft) [3] in the mud along the banks of the 4,350 km (2,703 mi)
Mekong River in Southeast Asia.
From front to back, the basic shape of the earthworm is a cylindrical tube, divided into a series of
segments (called metamerisms) that compartmentalize the body. Furrows are generally externally visible
on the body demarking the segments; dorsal pores and nephridiopores exude a fluid that moistens and
protects the worm's surface, allowing it to breathe. Except for the mouth and anal segments, each segment
carries bristle-like hairs called lateral setae used to anchor parts of the body during movement; species
may have four pairs of setae on each segment or more than eight sometimes forming a complete circle of
setae per segment. Special ventral setae are used to anchor mating earthworms by their penetration into
the bodies of their mates.
Generally, within a species, the number of segments found is consistent across specimens, and individuals
are born with the number of segments they will have throughout their lives. The first body segment
(segment number 1) features both the earthworm's mouth and, overhanging the mouth, a fleshy lobe
called the prostomium, which seals the entrance when the worm is at rest, but is also used to feel and
chemically sense the worm's surroundings. Some species of earthworm can even use the prehensile
prostomium to grab and drag items such as grasses and leaves into their burrow.
An adult earthworm develops a belt-like glandular swelling, called the clitellum, which covers several
segments toward the front part of the animal. This is part of the reproductive system and produces egg
capsules. The posterior is most commonly cylindrical like the rest of the body, but depending on the
species, may also be quadrangular, octagonal, trapezoidal, or flattened. The last segment is called
the periproct; the earthworm's anus, a short vertical slit, is found on this segment [52] [53].
The exterior of an individual segment is a thin cuticle over skin, commonly pigmented red to brown,
which has specialized cells that secrete mucus over the cuticle to keep the body moist and ease movement
through soil. Under the skin is a layer of nerve tissue, and two layers of muscles—a thin outer layer of
circular muscle, and a much thicker inner layer of longitudinal muscle. [7] Interior to the muscle layer is a
fluid-filled chamber called a coelom that by its pressurization provides structure to the worm's boneless
body. The segments are separated from each other by septa (the plural of "septum") which are perforated
transverse walls, allowing the coelomic fluid to pass between segments. A pair of structures
called nephrostomes are located at the back of each septum; a nephric tubule leads from each
nephrostome through the septum and into the following segment. This tubule then leads to the main body
fluid filtering organ, thenephridium or metanephridium, which removes metabolic waste from
the coelomic fluid and expels it through pores called nephridiopores on the worm's sides; usually two
nephridia (sometimes more) are found in most segments.At the center of a worm is the digestive tract,
which runs straight through from mouth to anus without coiling, and is flanked above and below by blood
vessels (the dorsal blood vessel and the ventral blood vessel as well as a subneural blood vessel) and
the ventral nerve cord, and is surrounded in each segment by a pair of pallial blood vessels that connect
the dorsal to the subneural blood vessels.
Many earthworms can eject coelomic fluid through pores in the back in response to stress;
Australian Didymogaster sylvaticus (known as the "blue squirter earthworm") can squirt fluid as high as
30 cm (12 in) [54], [55].
Experiments:
We had to take weight of Albendazole of 700 mg, 500 mg, 400 mg, 350 mg, 300 mg and Mebendazole of
700 mg, 400 mg, 350 mg, 300 mg, 200 mg. We did experiment on six formulations, each formulation
contained total 700 mg API. We took 35 ml water for each formlation for obtaining a density of 20
mg/ml. Dimethylformamide(DMF) was used to dissolve Albendazole and Mebendazole in water.The six
formlations,
1. Combination 1- (400 mg Albendazole + 300 mg Mebendazole)
5. Combination 5- (700 mg Albendazole)
6. Combination 6- (700 mg Mebendazole)

Figure 3.2: Different Formulations
Figure 3.3: Dead Earthworms after Experiment
3.3.2. Different Methods for formulation: Different methods can be used to formulate the combination
of Albendazole and Mebendazole [56].
Non Aqueous Granulation: Molecules that need wet granulation are those not suited for

dry granulation process – high dose, poor flow, low in bulk density, without binding properties.
Depending on molecule sensitivity, non-aqueous(organic) solvents are used for the granulation process.
Aqueous Granulation: The process of adding a liquid solution to powders involves the massing of a
mix of dry primary powder particles using a granulating fluid. The fluid contains a solvent that must be
volatile, so that it can be removed by drying, and be non-toxic. Aqueous granulation forms by binding the
powders together with an adhesive, instead of by compaction. bridges are developed between the particles
and the tensile strength of bonds increases as the amount of liquid added is increased [57].
Step 1: Weighing and mixing of formulation ingredients (excluding the lubricant).
Step 2: Preparing the damp mass.
Step 3: Wet screening/ Screening the dampened powder into pellets or granules.
Step 4: Drying of moist granules.
Step 5: Sizing the granulation by dry screening

Step 6: Lubrication of granules
Step 7: Compression of granules into tablets
Direct Compression: The term “direct compression” is defined as the process by which tablets
are compressed directly from powder mixture of API and suitable excipients. No pretreatment of the
powder blend by wet or dry granulation procedure is required. The processing of drug with excipients can
be achieved without any need of granulation and related unit operations. By simply mixing in a blender,
formulation ingredients can be processed and compressed into tablets without any of the ingredients
having to be changed. Direct compression method requires fewer processing steps (unit operations) and
less equipment. Therefore, the method is potentially less expensive than other methods used in tablet
manufacture. High-dose drugs may present problems with direct compression if it is not easily
compressible by itself. The choice of excipients used in the manufacture of tablets by direct compression
technology is highly restricted since most materials do not have inherent binding properties.
3.3.3. Method Used
Aqueous Granulation: All the ingredients were separately weighed and sifted using mesh no. 40.
Albendazole, Mebendazole, Lactose monohydrate, Starch, and Sodium starch glycolate were mixed for
ten minutes.
For the Preparation of binder dispersion, purified water was taken in a beaker,stirred with a glass rod to
disperse starch until no lumps were observed.
Then the above dry mixture was granulated with binder solution and dried in the tray drier at the
temperature of 40-50ºC until the moisture reduces down to NMT-2%. The dried granules were passed
through mesh no. 30.
Then Mannitol was passed through mesh no. 30, Sodium saccharine,orange flavor and color were passed
through mesh no.100.All these were then added to the dried granules and blended for ten minutes.
Finally, the above blend was lubricated with Magnesium stearate, Talc, Aerosil for two minutes.
The mixture was compressed in a rotary tablet machine in order to formulate the combination tablet.
With this method 6 formulations are made.
Formulations:
1.Formulation A
Table 3.1: List of Different Ingredients Used in Formulation A
Materials Weights
Albendazole 400 mg
Mebendazole 300 mg
Lactose 200 mg
Starch 37 mg
Sodium Starch Glycolate 66 mg
Starch for binder 34 mg
Mannitol 134 mg
Sodium Saccharin 13 mg
Magnesium Stearate 8 mg
Talc 8 mg
Aerosil Not significant
Orange Color Not significant
Orange Flavour Not significant
Method A: Albendazole 400 mg, Mebendazole 300 mg, Lactose monohydrate 200 mg, Starch 37 mg,
and Sodium starch glycolate 66 mg were mixed for ten minutes for each tablet.
disperse starch 34 mg until no lumps were observed.
Then Mannitol 134 mg was passed through mesh no. 30, Sodium saccharine 13 mg, orange flavor and
color were passed through mesh no.100.All these were then added to the dried granules and blended for
ten minutes. Finally, the above blend was lubricated with Magnesium stearate 8 mg, Talc 8 mg, Aerosil
for two minutes.
2. Formulation B:
Table 3.2: List of Different Ingredients Used in Formulation B
Materials Weights
Albendazole 400 mg
Mebendazole 300 mg
Lactose 200 mg
Starch 35 mg
Mannitol 134 mg
Talc 8 mg
Method B: Albendazole 400 mg, Mebendazole 300 mg, Lactose monohydrate 200 mg, Starch 35 mg, and
Sodium starch glycolate 68 mg were mixed for ten minutes for each tablet.
for two minutes.
3. Formulation C:
Table 3.3: List of Different Ingredients Used in Formulation C
Materials Weights
Albendazole 400 mg
Mebendazole 300 mg
Lactose 200 mg
Starch 33 mg
Mannitol 134 mg
Talc 8 mg
Method C: Albendazole 400 mg, Mebendazole 300 mg, Lactose monohydrate 200 mg, Starch 33 mg,
for two minutes.
4. Formulation D:
Table 3.4: List of Different Ingredients Used in Formulation D
Materials Weights
Albendazole 400 mg
Mebendazole 300 mg
Lactose 200 mg
Starch 31 mg
Mannitol 134 mg
Talc 8 mg
Method D: Albendazole 400 mg, Mebendazole 300 mg, Lactose monohydrate 200 mg, Starch 31 mg,
for two minutes.
Formulation E:
Table 3.5: List of Different Ingredients Used in Formulation E
Materials Weights
Albendazole 400 mg
Mebendazole 300 mg
Lactose 200 mg
Starch 29 mg
Mannitol 134 mg
Talc 8 mg
Method E: Albendazole 400 mg, Mebendazole 300 mg, Lactose monohydrate 200 mg, Starch 29 mg, and
for two minutes.
Formulation F:
Table 3.6: List of Different Ingredients Used in Formulation F
Materials Weights
Albendazole 400 mg
Mebendazole 300 mg
Lactose 200 mg
Starch 27 mg
Mannitol 134 mg
Talc 8 mg
Method F: Albendazole 400 mg, Mebendazole 300 mg, Lactose monohydrate 200 mg, Starch 29 mg, and
disperse starch 34 mg until no lumps were observed. Then the above dry mixture was granulated with
binder solution and dried in the tray drier at the temperature of 40-50ºC until the moisture reduces down
to NMT-2%. The dried granules were passed through mesh no. 30.
for two minutes.
3.3.4. Qualitative Testing:
3.3.4.1. Carr’s Index:
Carr's index is an indication of the compressibility of a powder.
Carr's Compressibility Index is named after the scientist Ralph J. Carr, Jr.
' Tapped density−Bulk density
The Carr index is calculated by the formula, Car r s Index = ×100 %
Tapped density
where is the freely settled bulk density of the powder, and is the tapped bulk density of the powder after
"tapping down".
Figure 3.4: Bulk Volume Figure 3.5: Tapped Volume
The Carr’s index is frequently used in pharmaceutics as an indication of the flowability of a powder. In a
free-flowing powder, the bulk density and tapped density would be close in value, therefore, the Carr’s
index would be small. On the other hand, in a poor-flowing powder where there are greater interparticle
interactions, the difference between the bulk and tapped density observed would be greater, therefore, the
Carr index would be larger. A Carr’s index greater than 25 is considered to be an indication of poor
flowability, below 15 is considered to be an indication of good flowability [58].
3.3.4.2. Hausner Ratio:

The Hausner ratio is a number that is correlated to the flowability of a powder or granular material. It is
named after the engineer Henry H. Hausner (1900–1995).
The Hausner ratio is calculated by the formula,
Tapped density
Hausner ratio=
Bulk density
The Hausner ratio is not an absolute property of a material; its value can vary depending on the
methodology used to determine it.
The Hausner ratio is used in a wide variety of industries as an indication of the flowability of a powder. A
Hausner ratio greater than 1.25 is considered to be an indication of poor flowability. The Hausner ratio
(H) is related to the Carr’s index(C), another indication of flowability . Both the Hausner ratio and the
Carr index are sometimes criticized, despite their relationships to flowability being established
empirically, as not having a strong theoretical basis [59]. Use of these measures persists, however,
because the equipment required to perform the analysis is relatively cheap and the technique is easy to
learn.
3.3.4.3. Angle Of Repose:
The angle of repose, or critical angle of repose, of a granular material is the steepest angle of descent or

dip relative to the horizontal plane to which a material can be piled without slumping. At this angle, the
material on the slope face is on the verge of sliding. The angle of repose can range from 0° to 90°.
Figure 3.6: Angle of repose
The equation for calculating the angle of repose is: tan-1(2h/d). Using your scientific calculator, multiply

height by 2 and divide this value by the distance.
The size of the particles is a factor. Other factors being equal, fine grained material will form a shallower
pile, with a smaller angle of repose than coarser grains. Moisture affects the angle of repose, as anyone
who has ever built a sand castle can confirm [60].
 Tilting box method. This method is appropriate for fine-grained, non-cohesive materials, with

individual particle size less than 10 mm.
 Fixed funnel method. The material is poured through a funnel to form a cone.
 Revolving cylinder method. The material is placed within a cylinder with at least one
transparent face.
Method Used: Fixed Funnel Method.
3.3.4.4. Hardness Test:
A hardness test is a method employed to measure the hardness of a material. Hardness refers to a

material's resistance to permanent indentation. There are numerous techniques to measure hardness and
each of these tests can identify varying hardness values for a single material under testing.
Figure 3.7: Hardness Test Machine
Hardness is a characteristic of a material, not a fundamental physical property. It is defined as the

resistance to indentation, and it is determined by measuring the permanent depth of the indentation.
Considerations:
1.Material
2.Sample Size
3.Thickness
4.Scale
5.Shape
6.Gage R & R
Material
The type of material and expected hardness will determine test method. Materials such as hardened
bearing steels have small grain size and can be measured using the Rockwell scale due to the use of
diamond indenters and high PSI loading. Materials such as cast irons and powder metals will need a much
larger indenter such as used with Brinell scales. Very small parts or small sections may need to be
measured on a microhardness tester using the Vickers or Knoop Scale.
When selecting a hardness scale, a general guide is to select the scale that specifies the largest load and
the largest indenter possible without exceeding defined operation conditions and accounting for
conditions that may influence the test result.
Sample size
The smaller the part, the lighter the load required to produce the required indentation. On small parts, it is
particularly important to be sure to meet minimum thickness requirements and properly space
indentations away from inside and outside edges. Larger parts need to be fixtured properly to ensure
secure placement during the test process without the chance for movement or slippage. Parts that either
overhang the anvil or are not easily supported on the anvil should be clamped into place or properly
supported.
Cylindrical samples
A correction to a test result is needed when testing on cylinder shapes with small diameters due to a
difference between axial and radial material flow. Roundness correction factors are added to your testing
result based on the diameter of convex cylinder surfaces. Additionally, it is important to maintain a
minimum spacing equal to 2~1/2 times the indentation's diameter from an edge or another indentation.
Sample Thickness
Your sample should have a minimal thickness that is at least 10x (ten times) the indentation depth that is
expected to be attained. There are minimum, allowable thickness recommendations for regular and
superficial Rockwell methods.

Scales
Sometimes it is necessary to test in one scale and report in another scale. Conversions have been
established that have some validity, but it is important to note that unless an actual correlation has been
completed by testing in different scales, established conversions may or may not provide reliable
information. Refer to ASTM scale conversion charts for non-austenitic metals in the high hardness
range and low hardness range. Also refer to ASTM standard E140 for more scale conversion
information.
Gage R&R
Gage Repeatability and Reproducibility Studies were developed to calculate the ability of operators and
their instruments to test accordingly within the tolerances of a given test piece. In hardness testing, there
are inherent variables that preclude using standard Gage R&R procedures and formulas with actual test
pieces. Material variation and the inability to retest the same area on depth measuring testers are two
significant factors that affect GR&R results. In order to minimize these effects, it is best to do the study
on highly consistent test blocks in order to minimize these built-in variations.
Newage Testing Instruments hardness testers operate are ideally suited for these studies. Unfortunately,
since these studies can only be effectively done on test blocks, their value does not necessarily translate
into actual testing operations. There are a host of factors that can be introduced when testing under real
conditions. Newage testers excel at testing in real-world conditions by reducing the effects of vibration,
operator influence, part deflection due to dirt, scale, a specimen flexing under load.
Types of Hardness Test:
 Brinell Hardness Test.
 Rockwell Hardness Test.
 Rockwell Superficial Hardness Test.
 Vickers Hardness Test.
 Knoop Hardness Test.
 Shore Scleroscope Hardness Test.
Brinell Hardness Test:

Figure 3.8: Brinell Hardness Test
In this test a hardened steel ball of 2.5, 5 or 10 mm in diameter is used as indenter.
The loading force is in the range of 300N to 30000N (300N for testing lead alloys, 5000N for testing
aluminum alloys, 10000N for copper alloys, 30000N for testing steels). The Brinell Hardness Number
(HB) is calculated by the formula:
HB = 2F/ (3.14D*(D-(D² - Di²)½))
Where
F- applied load, kg
D – indenter diameter, mm
Di – indentation diameter, mm.
In order to eliminate an influence of the specimen supporting base, the specimen should be seven times
(as minimum) thicker than indentation depth for hard alloys and fifteen times thicker than indentation
depth for soft alloys [61].
Rockwell Hardness Test:
Figure 3.9: Rockwell Hardness Test

Figure 3.10: Rockwell Hardness Test
In the Rockwell test the depth of the indenter penetration into the specimen surface is measured. The
indenter may be either a hardened steel ball with diameter 1/16”, 1/8” or a spherical diamond cone of
120º angle (Brale).
Loading procedure starts from applying a minor load of 10 kgf (3kgf in Rockwell Superficial Test) and
then the indicator, measuring the penetration depth, is set to zero. After that the major load (60, 100 or
150 kgf)is applied. The penetration depth is measured after removal of the major load [62].
Hardness is measured in different scales (A, B, C, D, E, F, G, H, K) and in numbers, having no units (in
contrast to Brinell and Vickers methods).
Aluminum alloys, copper alloys and soft steels are tested with 1/16” diameter steel ball at 100 kgf load
(Rockwell hardness scale B).
Harder alloys and hard cast iron are tested with the diamond cone at 150 kgf ( Rockwell hardness scale
C).
An example of Rockwell test result: 53 HRC. It means 53 units, measured in the scale C by the method
HR (Hardness Rockwell).
Rockwell Superficial Hardness Test

Rockwell Superficial Test is applied for thin strips, coatings, carburized surfaces.
Reduced loads (15 kgf, 30 kgf, and 30 kgf) as a major load and deduced preload (3kgf) are used in the
superficial test.
Depending on the indenter, two scales of Rockwell Superficial method may be used: T (1/16” steel ball)
or N (diamond cone).
62 R30T means 62 units, measured in the scale 30T (30 kgf, 1/16” steel ball indenter) by the Rockwell
Superficial test [63].
Figure 3.11: Rockwell Superficial Hardness Test
Vickers Hardness Test:
The principle of the Vickers Hardness method is similar to the Brinell method.
The Vickers indenter is a 136 degrees square-based diamond pyramid.
The impression, produced by the Vickers indenter is clearer, than the impression of Brinell indenter,
therefore this method is more accurate [64], [65].
The load, varying from 1kgf to 120 kgf, is usually applied for 30 seconds.
The Vickers number (HV) is calculated by the formula:
HV = 1.854*F/ D²
Where
F-applied load, kg
D – length of the impression diagonal, mm
The length of the impression diagonal is measured by means of a microscope, which is usually an integral
part.
Figure 3.12: Vickers Hardness Test
Knoop Hardness Test:

Figure 3.13: Knoop Hardness Test
A diamond pyramid indenter with angles 130º and 170º30’ is used in this method.
The Knoop Hardness Test is applied for testing soft material and thin coating, since the penetration depth
is very small (about 1/30 of the impression length).
The loading force in the Knoop method are usually in the range of 10 gf to 1000gf (micro-hardness range)
[66].
The Knoop number (HK) is calculated by the formula:
HK = 14.229*F/L²
Where
F-applied load, kg
L – long diagonal of the impression, mm
Shore Scleroscope Hardness Test:

The Shore Scleroscope hardness is associated with the elasticity of the material.
The appliance consists of a diamond-tipped hammer, falling in a graduated glass tube from a definite
height. The tube is divided into 140 equal parts.
The height of the first rebound is the hardness index of the material.
The harder the material, the higher the rebound.
The Shore method is widely used for measuring hardness of large machine components like rolls, gears,
dies, etc.
The Shore scleroscope is not only small and mobile, it also leaves no impressions on the tested surface.
Method Used: Manual Hardness testing method
3.3.4.5. Dissolution Test:
Dissolution testing is an in vitro method that characterizes how an API is extracted out of a solid dosage
form. It can indicate the efficiency of in vivo dissolution but does not provide any information on drug
substance absorption.
When administered through oral route, the drug must be absorbed from the GI tract, and for the
absorption the drug should be in solution form, thus assessment of dissolution characteristics. nature of
the solvent including its pH and temperature, and stirring and mixing environment.
In the pharmaceutical industry, drug dissolution testing is routinely used to provide critical in vitro drug
release information for both quality control purposes, i.e., to assess batch-to-batch consistency of solid
oral dosage forms such as tablets, and drug development, i.e., to predict in vivo drug release profiles .
Factors affecting dissolution

A dissolution test measures the amount of drug that goes into solution over a period of time under
standardized conditions. Factors that affect the dissolution of a drug product include the intrinsic
properties of the API (e.g., solubility, wettability, particle size, surface area, morphology, polymorphs),
the formulation composition and characteristics (e.g., excipients, hardness, manufacturing process), and
the dissolution method used for its assessment (e.g., apparatus, medium, test conditions, sampling, and
sample analysis) .
Method development
In dissolution testing, the aim is to develop a discriminatory method that is sensitive to variables that
affect the dissolution rate, and consequently, the in-vivo performance of the drug product. The method
must be able to distinguish between drug products manufactured under target conditions and formulations
with meaningful variations for the most relevant critical manufacturing variables, such as drug substance
particle size, compression force, and tablet hardness, for example. The dissolution method should also be
sufficiently rugged and reproducible for daily operations as well as transferable between laboratories [67]
Dissolution apparatus
The United States Pharmacopeia and European Pharmacopoeia describe four different dissolution
apparatuses that can be used to develop an appropriate dissolution method for oral solid-dosage forms
based on the drug product characteristics .
Apparatus 1 (basket) and Apparatus 2 (paddle) are most commonly used methods in dissolution
testing. Apparatus 1 consists of a vessel made of glass or other inert, transparent material and a cylindrical
basket attached to the lower part of a rotating stirrer. The set up for Apparatus 2 is generally the same
except that a paddle formed from a blade and a shaft is used as the stirring element. Sinkers may be used
for dosage forms that would otherwise float. These apparatuses are simple, robust, well standardized, and
flexible enough to allow dissolution testing for a wide variety of oral solid-dosage forms. Baskets and
paddles are recommended for dissolution method development unless they have been shown to be
unsatisfactory (1).
Figure 3.14: Dissolution Test
Apparatus 3 (reciprocating cylinder) was develop in recognition of the need for a system that
sequentially alters the dissolution conditions to mimic the gastrointestinal tract, so that in vitro-in
vivo correlations can be established. Apparatus 3 consists of a set of cylindrical, flat-bottomed glass
vessels, a set of glass reciprocating cylinders with inert fittings and screens at the top and bottom of the
cylinders, and a motor and drive assembly to reciprocate the cylinders vertically inside the vessels; and if
desired, the reciprocating cylinders can be moved horizontally to different rows of vessels (8, 9). Unlike
the baskets and paddles, agitation in Apparatus 3 comes from dipping within the vessel, rather than
through a stirred media approach. The advantages of the reciprocating cylinder include the ability for pH
profiling, which enables simulation of pH changes in the gastrointestinal tract; and the system can be
programmed to run dissolution in different media and at different speeds at various times (i.e.,
programmable dip speeds at each interval and programmable interval time). Such flexibility allows for
better in vitro-in vivo modeling.
Apparatus 4 (flow-through cell) consists of a reservoir containing the dissolution medium, a pump that
forces the medium upwards through the vertically positioned flow-cell, and a water bath to maintain the
temperature of the dissolution medium (8, 9). The flow-through cell apparatus can operate in two
different modes—an open system with continuous flow of solvent passing through the cell from the
reservoir, and a closed system where a fixed volume of media is recirculated. The open system is suitable
for poorly soluble drugs, which require a high volume of media for dissolution. The closed system, on the
other hand, is used when a low volume of medium is required.
Method Used: In dissolution test Apparatur 2 with paddle was used. The filtrates at o, 5, 10, 15 minutes
were used in UV Spectroscopy at 308 nm and 234 nm.
3.3.4.6. Disintegration Test:
This test is provided to determine whether tablets or capsules disintegrate within the prescribed time
when placed in a liquid medium under the experimental conditions presented below.
Figure 3.15: Disintegration Test
For the purposes of this test disintegration does not imply complete dissolution of the unit or even of its
active constituent. Complete disintegration is defined as that state in which any residue of the unit, except
fragments of insoluble coating or capsule shell, remaining on the screen of the test apparatus or adhering
to the lower surface of the discs, if used, is a soft mass having no palpably firm core.
Use apparatus A for tablets and capsules that are not greater than 18 mm. For larger tablets and capsules
use apparatus B.
Test A. Tablets and capsules of normal size
This text is based on the internationally-harmonized texts developed by the Pharmacopoeial

Discussion Group (PDG). Some editorial modifications have been made in order to be in line with
the style used in The International Pharmacopoeia.
Apparatus. The apparatus consists of a basket-rack assembly, a 1000 mL, low-form beaker, 138–160
mm in height and having an inside diameter of 97–115 mm for the immersion fluid, a thermostatic
arrangement for heating the fluid between 35 °C and 39 °C and a device for raising and lowering the
basket in the immersion fluid at a constant frequency rate between 29 and 32 cycles per minute, through a
distance of not less than 53 mm and not more than 57 mm. The volume of the fluid in the vessel is such
that at the highest point of the upward stroke the wire mesh remains at least 15 mm below the surface of
the fluid and descends to not less than 25 mm from the bottom of the vessel on the downward stroke. At
no time should the top of the basket-rack assembly become submerged. The time required for the upward
stroke is equal to the time required for the downward stroke and the change in stroke direction is a
smooth transition rather than an abrupt reversal of motion. The basket-rack assembly moves vertically
along its axis. There is no appreciable horizontal motion or movement of the axis from the vertical.
Basket-rack assembly . The basket-rack assembly consists of six open-ended transparent tubes each
75.0–80.0 mm long and having an internal diameter of 20.70–23.00 mm and a wall 1.0–2.8 mm thick; the
tubes are held in a vertical position by two plates, each 88–92 mm in diameter and 5.00–8.50 mm in
thickness, with six holes, each 22–26 mm in diameter, equidistant from the centre of the plate and equally
spaced from one another. Attached to the lower surface of the lower plate is a woven stainless steel wire
mesh which has a plain square weave with 1.8–2.2 mm apertures and with a wire diameter of 0.570–
0.660 mm. The parts of the apparatus are assembled and rigidly held by means of three bolts passing
through the two plates. A suitable means is provided to suspend the basket-rack assembly from the
raising and lowering device using a point on its axis.
The design of the basket-rack assembly may be varied somewhat provided the specifications for the
glass tubes and the screen mesh size are maintained. The basket-rack assembly conforms to the
dimensions.
Discs. The use of discs is permitted only where specified or allowed. Each tube is provided with a
cylindrical disc 9.35–9.65 mm thick and 20.55–20.85 mm in diameter. The disc is made of a suitable,
transparent plastic material having a specific gravity of 1.18–1.20. Five parallel 1.9–2.1 mm holes
extend between the ends of the cylinder. One of the holes is centered on the cylindrical axis. The other
holes are centered 5.8–6.2 mm from the axis on imaginary lines perpendicular to the axis and parallel to
each other. Four identical trapezoidal-shaped planes are cut into the wall of the cylinder nearly
perpendicular to the ends of the cylinder. The trapezoidal shape is symmetrical; its parallel sides
coincide with the ends of the cylinder and are parallel to an imaginary line connecting the centres of
two adjacent holes 6 mm from the cylindrical axis. The parallel side of the trapezoid on the bottom of
the cylinder has a length of 1.5–1.7 mm and its bottom edges lie at a depth of 1.50–1.80 mm from the
cylinder’s circumference. The parallel side of the trapezoid on the top of the cylinder has a length of
9.2–9.6 mm and its centre lies at a depth of 2.5–2.7 mm from the cylinder’s circumference. All surfaces
of the disc are smooth. If the use of discs is specified add a disc to each tube and operate the apparatus
as directed under procedure.
Procedure. Place one dosage unit in each of the six tubes of the basket and if specified add a disc.
Operate the apparatus using water as the immersion fluid unless another liquid is specified and maintain
its temperature at 35–39 °C. At the end of the specified time lift the basket from the fluid and observe
the dosage units: all of the dosage units have disintegrated completely. If one or two dosage units fail to
disintegrate repeat the test on 12 additional dosage units. The requirements of the test are met if not less
than 16 of the 18 dosage units tested are disintegrated.
Test B – Large tablets and large capsules
This test is reproduced from The European Pharmacopoeia with permission and with appropriate editorial
modifications.
Apparatus. The main part of the apparatus (Figure 2) is a rigid basket-rack assembly supporting 3
cylindrical transparent tubes 77.5 ± 2.5 mm long, 33.0 mm ± 0.5 mm in internal diameter and with a wall
thickness of 2.5 ± 0.5 mm. Each tube is provided with a cylindrical disc 31.4 ± 0.13 mm in diameter and
15.3 ± 0.15 mm thick, made of transparent plastic with a relative density of1.18–1.20. Each disc is
pierced by 7 holes, each 3.15 ± 0.1 mm in diameter, 1 in the centre and the other 6 spaced equally on a
circle of radius 4.2 mm from the centre of the disc. The tubes are held vertically by 2 separate and
superimposed rigid plastic plates 97 mm in diameter and 9 mm thick with 3 holes. The holes are
equidistant from the centre of the plate and equally spaced. Attached to the underside of the lower plate is
a piece of woven gauze made from stainless steel wire 0.63 ± 0.03 mm in diameter and having mesh
apertures of 2.0 ± 0.2 mm. The plates are held rigidly in position and 77.5 mm apart by vertical metal
rods at the periphery. A metal rod is also fixed to the centre of the upper plate to enable the assembly to
be attached to a mechanical device capable of raising and lowering it smoothly at a constant frequency of
between 29 and 32 cycles per minute, through a distance of 55 ± 2 mm.
The assembly is suspended in the specified liquid medium in a suitable vessel preferably a 1 litre beaker.
The volume of the liquid is such that when the assembly is in the highest position the wire mesh is at
least 15 mm below the surface of the liquid, and when the assembly is in the lowest position the wire
mesh is at least 25 mm above the bottom of the beaker and the upper open ends of the tubes remain
above the surface of the liquid. A suitable device maintains the temperature of the liquid at 35–39 °C
[68].
Method Used: Test 6 tablets or capsules either by using 2 basket-rack assemblies in parallel or by
repeating the procedure. In each of the tubes place 1 tablet or capsule and, if prescribed, add a disc;
suspend the assembly in the beaker containing the specified liquid. Operate the apparatus using water as
the immersion fluid unless another liquid is specified for the prescribed period, withdraw the assembly
and examine the state of the tablets or capsules. To pass the test all 6 of the tablets or capsuls must have
disintegrated.
3.3.4.7. Friability Test:
Friability (the condition of being Friable) testing is a method, which is employed to determine
physical strength of compressed and uncoated tablets upon exposure to mechanical shock and
attrition. In simple words, friability test tells how much mechanical stress tablets are able to
withstand during their manufacturing, distribution and handling by the customer. Throughout
pharmaceutical industry, friability testing has become an accepted technology and the instrument
used in to perform this process is called Friabilator or Friability Tester. The mechanical strength of
tablet or granules can be determined by its hardness and through friability test. The strength of a
tablet plays a very important role in its marketing and dissolution.
Figure 3.16 : Friability Test
This method text provides guidance for the friability determination of compressed, uncoated tablets. The
test procedure presented is generally applicable to most compressed tablets. Measurement of tablet
friability supplements other physical strength measurements, such as tablet breaking force.
Use a drum with an internal diameter between 283 and 291 mm and a depth between 36 and 40 mm, of
transparent synthetic polymer with polished internal surfaces, and subject to minimum static build-up (see
Figure 1 for a typical apparatus). One side of the drum is removable. The tablets are tumbled at each turn
of the drum by a curved projection with an inside radius between 75.5 and 85.5 mm that extends from the
middle of the drum to the outer wall. The outer diameter of the central ring is between 24.5 and 25.5 mm.
The drum is attached to the horizontal axis of a device that rotates at 25 ± 1 rpm. Thus, at each turn the
tablets roll or slide and fall onto the drum wall or onto each other. For tablets with a unit weight equal to
or less than 650 mg, take a sample of whole tablets n corresponding as near as possible to 6.5 g. For
tablets with a unit weight of more than 650 mg, take a sample of 10 whole tablets. The tablets should be
carefully dedusted prior to testing. Accurately weigh the tablet sample, and place the tablets in the drum.
Rotate the drum 100 times, and remove the tablets. Remove any loose dust from the tablets as before, and
accurately weigh.Generally, the test is run once. If obviously cracked, cleaved, or broken tablets are
present in the tablet sample after tumbling, the sample fails the test. If the results are difficult to interpret
or if the weight loss is greater than the targeted value, the test should be repeated twice and the mean of
the three tests determined. A maximum weight loss (obtained from a single test or from the mean of three
tests) of not more than 1.0% is considered acceptable for most products.If tablet size or shape causes
irregular tumbling, adjust the drum base so that the base forms an angle of about 10º with the horizontal
and the tablets no longer bind together when lying next to each other, which prevents them from falling
freely. Effervescent tablets and chewable tablets may have different specifications as far as friability is
concerned. In the case of hygroscopic tablets, an appropriate humidity-controlled environment is required
for testing. Drums with dual scooping projections, or apparatus with more than one drum, for the running
of multiple samples at one time, are also permitted [69].
3.3.4.8. FTIR Analysis:

Fourier Transform Infrared (FTIR) Spectroscopy is commonly referred to as FTIR Analysis or FTIR
Spectroscopy. This infrared spectroscopy method is used to identify organic, polymeric, and in some
cases, inorganic materials. The FTIR test relies on infrared light to scan samples and observe bond
properties.
Figure 3.17: FTIR Machine

FTIR analysis services can identify compounds and the general type of material being analyzed when
there are unknowns. This technique is used to assess the purity of some inorganic samples and is highly
reliable for identifying polymer composition.
FTIR ANALYSIS FROM CHEMISTRY SPECIALISTS

Laboratory Testing Inc. performs FTIR spectroscopy services with a Thermo Scientific Nicolet iS10 FT-
IR Spectrometer. The Lab is located in the Philadelphia, PA area of the United States and also specializes
in metals testing.
During testing, when infrared radiation is passed through a sample, some radiation is absorbed by the
sample and the rest passes through. Qualitative information about the sample is provided.
ANALYZING SAMPLES IN A VARIETY OF FORMS

Our FTIR testing lab can analyze solid, liquid, powder or thin film samples using the following methods:
 Solids placed on a crystal (ATR)
 Liquids placed between two sodium chloride plates (Nujol)
 Thin film placed in a cassette
 Powdered sample mixed with potasium bromide and placed in a pellet (KBr pellet)
THE TEST PROCESS

Before FTIR analysis begins, the sample is prepared for testing using either the attenuated total
reflectance (ATR), Nujol or other technique. Enough sample is required to obtain an absorption spectrum.
The FTIR Spectrometer generates a graph in the form of an absorbance spectra, which shows the unique
chemical bonds and the molecular structure of the sample material. This absorption spectrum will have
peaks representing components present. These absorbance peaks indicate functional groups (e.g. alkanes,
ketones, acid chlorides). Different types of bonds, and thus different functional groups, absorb infrared
radiation of different wavelengths.
The analytical spectrum is then compared in a reference library program to identify components or to find
a “best match” for unknown material using the cataloged spectra for known materials [70].
ADDITIONAL CHEMISTRY SERVICES FROM LTI

FTIR Spectroscopy complements the wide-range of instrumental and wet chemistry services performed at
LTI for material characterization, qualitative and quantitative element analysis and failure analysis on a
variety of materials including metals, powdered metals, ores, ferroalloys, composites and ceramics.
Laboratory Testing Inc. is fully capable of preparing test specimens for all analysis [71].
LTI CAPABILITIES
 Identification of organic, polymeric, and some inorganic materials, including sample
composition, additives, contaminants and unknown materials
 Qualitative and quantitative results
 Absorbance and transmittance information is available
 Library of reference spectra
3.3.4.9. SEM Analysis:
The scanning electron microscope (SEM) uses a focused beam of high-energy electrons to generate a
variety of signals at the surface of solid specimens. The signals that derive from electron-sample
interactions reveal information about the sample including external morphology (texture), chemical
composition, and crystalline structure and orientation of materials making up the sample.
Figure 3.18: SEM Machine
In most applications, data are collected over a selected area of the surface of the sample, and a 2-
dimensional image is generated that displays spatial variations in these properties. Areas ranging from
approximately 1 cm to 5 microns in width can be imaged in a scanning mode using conventional SEM
techniques (magnification ranging from 20X to approximately 30,000X, spatial resolution of 50 to 100
nm). The SEM is also capable of performing analyses of selected point locations on the sample; this
approach is especially useful in qualitatively or semi-quantitatively determining chemical compositions
(using EDS), crystalline structure, and crystal orientations (using EBSD). The design and function of the
SEM is very similar to the EPMA and considerable overlap in capabilities exists between the two
instruments [72].
3.3.4.10. HPLC Analysis:
High-performance liquid chromatography or high-pressure liquid chromatography (HPLC) is a

chromatographic method that is used to separate a mixture of compounds in analytical chemistry and
biochemistry so as to identify, quantify or purify the individual components of the mixture
The most important benefits gain from the uses of HPLC technique in the industrial and analytical field
that it is help in structure elucidation and quantitative determination of impurities and degradation
products in bulk drug materials and pharmaceutical formulations.
Compared to other chromatographic techniques, such as TLC, HPLC is extremely quick and efficient. It
uses a pump, rather than gravity, to force a liquid solvent through a solid adsorbent material, with
different chemical components separating out as they move at different speeds.
There are two elution types: isocratic and gradient. In the first type constant eluent composition is
pumped through the column during the whole analysis.
HPLC as a solution to efficiency problems
While all of these basic principles hold true for all chromatographic separations, HPLC was developed as
method to solve some of the shortcomings of standard liquid chromatography. Classic liquid
chromatography has several severe limitations as a separation method. When the solvent is driven by
gravity, the separation is very slow, and if the solvent is driven by vacuum, in a standard packed column,
the plate height increases and the effect of the vacuum is negated. The limiting factor in liquid
chromatography was originally the size of the column packing, once columns could be packed with
particles as small as 3 µm, faster separations could be performed in smaller, narrower, columns. High
pressure was required to force the mobile phase and sample through these new columns, and previously
unneeded apparatus was required to maintain reproducibility of results in this new instruments. The use of
high pressures in a narrow column allowed for a more effective separation to be achieved in much less
time than was required for previous forms of liquid chromatography
Figure 3.19: HPLC Machine
Types of HPLC:
1. Normal Phase HPLC
This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and non-
polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile phases are hexane,
methylene chloride, chloroform, diethyl ether, and mixtures of these.
Polar samples are thus retained on the polar surface of the column packing longer than less polar
materials.
2. Reverse Phase HPLC
The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as
mixtures of water and methanol or acetonitrile. It works on the principle of hydrophobic interactions
hence the more nonpolar the material is, the longer it will be retained.
3. Size-exclusion HPLC
The column is filled with material having precisely controlled pore sizes, and the particles are separated
according to its their molecular size. Larger molecules are rapidly washed through the column; smaller
molecules penetrate inside the porous of the packing particles and elute later.
4. Ion-Exchange HPLC

The stationary phase has an ionically charged surface of opposite charge to the sample ions. This
technique is used almost exclusively with ionic or ionizable samples.
The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the
longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are
used to control elution time.
Apparatus
Specialized apparatus is required for an HPLC separation because of the high pressures and low
tolerances under which the separation occurs. If the results are to be reproducible, then the conditions of
the separation must also be reproducible. Thus HPLC equipment must be of high quality; it is therefore
expensive.
Solvent
The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid components
whose respective concentrations are varied depending on the composition of the sample. As the solvent is
passed through a very narrow bore column, any contaminants could at worst plug the column, or at the
very least add variability to the retention times during repeated different trials. Therefore HPLC solvent
must be kept free of dissolved gases, which could come out of solution mid-separation, and particulates.
Column
In the HPLC column, the components of the sample separate based on their differing interactions with the
column packing. If a species interacts more strongly with the stationary phase in the column, it will spend
more time adsorbed to the column's adsorbent and will therefore have a greater retention time. Columns
can be packed with solids such as silica or alumina; these columns are called homogeneous columns. If
stationary phase in the column is a liquid, the column is deemed a bonded column. Bonded columns
contain a liquid stationary phase bonded to a sold support, which is again usually silica or alumina. The
value of the constant Cdescribed in the van Deemter equation is proportional, in HPLC, to the diameter of
the particles that constitute the column's packing material.
Pump
The HPLC pump drives the solvent and sample through the column. To reduce variation in the elution,
the pump must maintain a constant, pulse free, flow rate; this is achieved with multi-piston pumps. The
presence of two pistons allows the flow rate to be controlled by one piston as the other recharges.
A syringe pump can be used for even greater control of flow rate; however, the syringe pump is unable to
produce as much pressure as a piston pump, so it cannot be used in all HPLC applications.
Detector
The HPLC detector, located at the end of the column, must register the presence of various components of
the sample, but must not detect the solvent. For that reason there is no universal detector that works for all
separations. A common HPLC detector is a UV absorption detector, as most medium to large molecules
absorb UV radiation. Detectors that measure fluorescence and refractive index are also used for special
applications. A relatively new development is the combination of an HPLC separation with an NMR
detector. This allows the pure components of the sample to be identified and quantified by nuclear
magnetic resonance after having been separated by HPLC, in one integrated process.
Technique
Normal Phase vs. Reverse Phase
If the stationary phase is more polar than the mobile phase, the separation is deemed normal phase. If the
stationary phase is less polar than the mobile phase, the separation is reverse phase. In reverse phase
HPLC the retention time of a compound increases with decreasing polarity of the particular species. The
key to an effective and efficient separation is to determine the appropriate ratio between polar and non-
polar components in the mobile phase. The goal is for all the compounds to elute in as short a time as
possible, while still allowing for the resolution of individual peaks. Typical columns for normal phase
separation are packed with alumina or silica. Alkyl, aliphatic or phenyl bonded phases are typically used
for reverse phase separation.
Gradient Elution vs. Isocratic Elution
If the composition of the mobile phase remains constant throughout the HPLC separation, the separation
is deemed an isocratic elution. Often the only way to elute all of the compounds in the sample in a
reasonable amount of time, while still maintaining peak resolution, is to change the ratio of polar to non-
polar compounds in the mobile phase during the sample run. Known as gradient chromatography, this is
the technique of choice when a sample contains components of a wide range of polarities. For a reverse
phase gradient, the solvent starts out relatively polar and slowly becomes more non-polar. The gradient
elution offers the most complete separation of the peaks, without taking an inordinate amount of time. A
sample containing compounds of a wide range of polarities can be separated by a gradient elution in a
shorter time period without a loss of resolution in the earlier peaks or excessive broadening of later peaks.
However, gradient elution requires more complex and expensive equipment and it is more difficult to
maintain a constant flow rate while there are constant changes in mobile phase composition. Gradient
elution, especially at high speeds, brings out the limitations of lower quality experimental apparatus,
making the results obtained less reproducible in equipment already prone to variation. If the flow rate or
mobile phase composition fluctuates, the results will not be reproducible [73].
Applications
HPLC can be used in both qualitative and quantitative applications, that is for both compound
identification and quantification. Normal phase HPLC is only 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; it cannot separate inorganic ions (they can be separated by ion exchange
chromatography). It cannot separate polysaccharides (they are too hydrophilic for any solid phase
adsorption to occur), nor polynucleotides (they adsorb irreversibly to the reverse phase packing). Lastly,
incredibly hydrophobic compounds cannot be separated effectively by RPLC (there is little selectivity).
Aside from these few exceptions, RPLC is used for the separation of almost all other compound varieties.
RPLC can be used to effectively separate similar simple and aromatic hydrocarbons, even those that
differ only by a single methylene group. RPLC effectively separates simple amines, sugars, lipids, and
even pharmaceutically active compounds. RPLC is also used in the separation of amino acids, peptides,
and proteins. Finally RPLC is used to separate molecules of biological origin. The determination of
caffeine content in coffee products is routinely done by RPLC in commercial applications in order to
guarantee purity and quality of ground coffee. HPLC is a useful addition to an analytical arsenal,
especially for the separation of a sample before further analysis.

3. Materials and Methods

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3. Materials and Methods

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CHAPTER THREE

MARETIALS AND METHOD

3. Materials and Methods

3.1.2. Mebendazole: Mebendazole is methyl 5-benzoylbenzimidazole-2-carbamate,molecular formula of

3.1.3. Sodium Starch Glycolate: Sodium starch glycolate is the sodium salt of carboxymethyl

3.1.6. Mannitol: Mannitol (C6H8(OH)6) is used in pharmaceutical products as a sweeting agent, tablet

3.1.8. Magnessium Stearate: Magnesium stearate (Mg(C18H3502)2 or octadecanoic acid) is a solid,

3.1.9. Talc: Talc is a clay mineral composed of hydrated magnesium silicate with the chemical formula

In the pharmaceutical industry it is used as an anticaking agent to improve powder flow

3.2. Apparatus and Machine:

3.2.1. Beaker: Used in biological experiment.

3.2.2. Conical Flask: Used in dissolution test, biological experiment.

3.2.3. Round Bottom Flask: Used in HPLC analysis, biological experiment.

3.2.4. Glass Rod: Used in biological experiment and others.

3.2.5. Weighing Balance: Used to measure the weight of materials.

3.2.6. pH Meter: Used for preparing buffer.

3.2.7. Funnel: Used in dissolution test, for measuring angle of repose.

3.2.10. Hardness Tester Machine: Used to measure the hardness of tablets.

3.2.14. FTIR Analysis Machine: Used to perform FTIR analysis of powders.

Figure 3.1: Different Apparatuses

3.3.1. In vitro Anthelmintic Assay

Experiment before Formulation

An earthworm is a tube-shaped, segmented worm found in the phylum Annelida. They have a world-wide

1. Combination 1- (400 mg Albendazole + 300 mg Mebendazole)

2. Combination 2- (350 mg Albendazole + 350 mg Mebendazole)

3. Combination 3- (500 mg Albendazole + 200 mg Mebendazole)

4. Combination 4- (300 mg Albendazole + 400 mg Mebendazole)

5. Combination 5- (700 mg Albendazole)

6. Combination 6- (700 mg Mebendazole)

Figure 3.3: Dead Earthworms after Experiment

Non Aqueous Granulation: Molecules that need wet granulation are those not suited for

Step 1: Weighing and mixing of formulation ingredients (excluding the lubricant).

Step 2: Preparing the damp mass.

Step 4: Drying of moist granules.

Step 5: Sizing the granulation by dry screening

3.3.3. Method Used

With this method 6 formulations are made.

Table 3.1: List of Different Ingredients Used in Formulation A

Table 3.2: List of Different Ingredients Used in Formulation B

Table 3.3: List of Different Ingredients Used in Formulation C

Table 3.4: List of Different Ingredients Used in Formulation D

Table 3.5: List of Different Ingredients Used in Formulation E

Table 3.6: List of Different Ingredients Used in Formulation F

3.3.4. Qualitative Testing:

3.3.4.1. Carr’s Index:

Carr's index is an indication of the compressibility of a powder.

3.3.4.2. Hausner Ratio:

The Hausner ratio is calculated by the formula,

3.3.4.3. Angle Of Repose:

The angle of repose, or critical angle of repose, of a granular material is the steepest angle of descent or

The equation for calculating the angle of repose is: tan-1(2h/d). Using your scientific calculator, multiply

 Tilting box method. This method is appropriate for fine-grained, non-cohesive materials, with

Method Used: Fixed Funnel Method.

3.3.4.4. Hardness Test:

A hardness test is a method employed to measure the hardness of a material. Hardness refers to a

Hardness is a characteristic of a material, not a fundamental physical property. It is defined as the

Types of Hardness Test:

Brinell Hardness Test:

In this test a hardened steel ball of 2.5, 5 or 10 mm in diameter is used as indenter.

Rockwell Hardness Test:

Figure 3.9: Rockwell Hardness Test

Rockwell Superficial Hardness Test

Figure 3.11: Rockwell Superficial Hardness Test