evaluation of drug by organoleptic, microscopical, physical, chemical and biological methods. spectroscopical methods, chromatography, leaf constants, linear measurements.

1. DRUG
EVALUATION
1. Morphological evaluation
2. Microscopical evaluation
3. Physical evaluation
4. Chemical evaluation
5. Biological evaluation
PREPARED BY –
ZMS, IICP

2. DEFINITION
• Evaluation of drug defined as confirmation of its identity and
determination of its quality and purity and detection of
adulterants.
• Evaluation of crude drugs is necessary because of these reasons :
1. Geographical and biochemical variation in the drug
2. Effect of treatment and storage
3. Substitution and adulteration
 Methods for evaluation of crude drugs :
1. Morphological evaluation
2. Microscopical evaluation
3. Physical evaluation
4. Chemical evaluation
5. Biological evaluation

3. 1. MORPHOLOGICAL / ORGANOLEPTIC EVALUATION
• It refers to evaluation of drugs by colour, odour, taste, size, shape and
texture.
• Organoleptic evaluation means the study of drugs by using organs of
senses, like eye, skin-touch, taste buds, etc.
• Organoleptic analysis represents the simplest yet the most human form
of analysis.
A. Colour :
• Colour is to be examined under an artificial light source and or day light
(natural light).
• The colour of the sample should be compared with that of a reference.
• E.g. Senna leaves are fresh green in colour
• Digitalis leaves are dark green in colour
• Brown colour of Cinnamon
• Yellow / Orange colour of Turmeric

4. B. Odour :
• Slow and repeated inhalation of the material provides necessary
information of its odour.
• Where no distinct/characteristic odour is noticeable, crude drugs is to
be crushed using gentle pressure to verify exact odour.
• If the material is toxic or dangerous, then determination of its odour is
done by pouring a small quantity of boiling water on to the crushed
sample.
• Determine the strength of the odour like weak, distinct, strong and
characteristic.
• Then sensations like rancid, fruity, moldy, aromatic, etc.
• E.g. essential oil containing crude drugs have aromatic odour.
• Ergot, Vinca, Neem have disagreeable odour.
• Aromatic odour of umbelliferous fruits like Fennel, Cumin, Coriander,
Clove.
• Strong aroma of Rose, Saffron and Vanilla.

5. C. Taste :
• Non toxic crude drugs can be tasted, while toxic drugs like Nux-vomica and
aconite should not be tasted.
• E.g. most of the alkaloid containing drugs are bitter in taste.
• Amla and Citrus fruits have Sour taste.
• Liquorice. Honey have sweet taste.
• Senna leaves, Linseed have mucilaginous taste.
D. Size and Shape :
• The length, width and thickness of the crude drugs are important while
evaluating a crude drug.
• E.g. width of Indian senna is smaller than Alexandrian senna.
• Wavy (snake) shape of Rauwolfia root.
• Disc shape of Nux-vomica seed.
• Flat surface of Linseed.
• Heart shape leaves of Digitalis.
• Human like shape of Ginseng roots and Ashwagandha.
• Star shape of Star anise.

6. • Shape of Bark :
• Bark is protective outer covering of Trunk, Branches and roots.
• Flat – Arjuna bark
• Curved – Cassia bark
• Channeled – Cinchona bark
• Re-curved – Kurchi bark
• Quill – Cascara bark
• Double quill – Cinnamon bark
• Extra features :
• Touch of the material describes its softness and hardness.
• The texture is best examined by taking a small quantity of material and
rubbing it between the thumb and finger.
• Texture usually described as smooth and rough.

7. Microscopical evaluation

8. • Every plant possesses a characteristic tissue
structure, which can be demonstrated through
study of tissue arrangements, cell walls and
reactions with stain and reagents.
• Microscopical evaluation involves histological study
of type and arrangement of tissues, presence of
characteristic features, such as stomata, starch
grains, calcium oxalate crystals, trichomes by using
magnification power of microscope.
• Qualitative microscopical studies utilizes different
stains to differentiate and identify different
microscopical characters.

9. • Examples :
• Iodine stains starch grains blue.
• Phloroglucinol and HCl for Lignin, which stains pink.
• Sudan red-III for oil detection, which stains red.
• Ferric chloride for tannins- black/blue/brown
• Ruthenium red for mucilage- pink color
• Chloral hydrate solution is useful to remove
chlorophyll.

12. A. Stomata :
• Stomata is a minute epidermal opening present on
aerial parts of the plants, having a central pore and
two kidney shaped similar cells known as guard
cells, which covered by varying number of
epidermal cells known as Subsidiary cells.
• Function : gaseous exchange & Transpiration

13. • Types of stomata :
1. Paracytic : guard cells covered by two subsidiary
cells, which are parallel to guard cells.
• E.g. Senna, Coca leaves
2. Diacytic : guard cells covered by two subsidiary
cells, which is at right angle to guard cells.
• E.g. Mint leaves, Vasaka leaves
3. Anisocytic stomata : two guard cells are covered
by three subsidiary cells.
• E.g. Datura leaves

14. 4. Anomocytic stomata : two guard cells are
covered by varying number of epidermal cells.
• E.g. Digitalis leaves, Neem leaves
5. Actinocytic stomata : two guard cells are
surrounded by a circle of radiating cells.
• E.g. Pilocarpus leaves

16. B. Trichomes
• Trichomes are the tubular elongated or glandular
outgrowth of the epidermal cell.
• Trichomes are also known as plant hairs.
• Present in the most of the part of plants, such as
leaves – digitalis, senna, seeds- nux vomica, fruit-
ladies finger
• Function : excrete water and volatile oil in case of
peppermint
• Present in most of the aerial parts of the plant but
absent in root.

17. • Types of trichomes :
1. Covering trichomes :
• Unicellular – Cannabis,
• Nux vomica – Lignified trichomes
• Multicellular – Tulsi
• Unbranched – Vasaka
• Branched – Rosemary
• Collapsed- Digitalis
2. Glandular trichomes :
• Datura

19. C. Calcium oxalate crystals :
• Several inorganic components like calcium oxalate
crystals, calcium carbonate crystals and silica are
frequently found in plants.
• They are crystalline in nature.
• Specific shape of calcium oxalate crystals can be used
for the identification of drugs.
• Due to this reason they are known as diagnostic
characters of the plant.
• Function : protein metabolism gives oxalic acid, which
is harmful to plants.
• To remove harmful effect of oxalic acid, plant forms
harmless calcium oxalate crystals with calcium ion.
(obtained from soil).

20. • Properties :
 Deposited in different tissues, in different forms
 Harmless to plant.
 Doesn’t take part in metabolism, hence called excretory
product.
• Significance:
1)They give protection to the plant against birds and animals.
2)They have great diagnostic value.
3)Presence and absence of crystals, type of crystals and
dimensions are useful in correct identification of crude drugs.
4)Help in detection of adulterants.
 Clove stalk contains calcium oxalate prisms but clove flower
bud does not.

22. • LINEAR MEASUREMENTS :
• linear measurements include size of starch grains,
length and width of fibers and trichomes.
• Diameter of starch grains present in Cassia bark, is
different from starch grain present in Cinnamon
bark.
• Measurement of length and width of fibers in
Cinchona bark is important characteristic for
evaluation of Cinchona.

24. Leaf constants
1. Stomatal number
2. Stomatal index
3. Vein islet number
4. Vein termination number
5. Palisade ratio

25. 1. Stomatal number :
• The stomatal number is average number of stomata per sq. mm
of epidermis of the leaf.
2. Stomatal index :
• Is the percentage of number of stomata forms to the total
number of epidermal cells.
S
S.I. =------------- x 100
E + S
Where, S.I. = Stomatal Index
S = Number of stomata per unit area
E = Number of epidermal cells in the same unit area

26. 3. Vein islet number :
• Number of vein islets per sq. mm of the leaf surface
4. Vein termination number :
• Number of vein terminations per sq. mm of leaf
surface
5. Palisade ratio:
 Palisade parenchyma tissue consists of row of
closely packed elongated cells with their longer
axis perpendicular to the upper epidermis.
 Palisade ratio is defined as average number of
palisade cells beneath each epidermal cell.

28. Quantitative microscopy
• Lycopodium spore method :
• Lycopodium is composed of the spores of Lycopodium
clavatum. Each spore is tetrahedral in shape, the base is
rounded and three flat side meet to form apex.
• Lycopodium spores are uniform in shape, appearance and
size (25 µm).
• On an average, 94000 spores per mg of powdered
lycopodium are present.
• By this figure one can calculate the weight of any number
of spores under any condition under the microscope.
• If the lycopodium has been fixed with a definite
proportion of another substance, one can find immediately
how much of the second substance has been added, when
examined microscopically.

29. • Procedure:
 A weighed quantity of the Lycopodium powder (spores)
and crude drug powder is mixed and suspended in viscous
liquid.
 A drop of this suspension is mounted on the slide and
examined under microscope with 4 mm objective.
 Lycopodium spores and number of particles like starch
grains or pollen grains are counted in 25 different areas.
 Counting is repeated 4 times with fresh mounting side.
 The number of pollen grains in 1 mg of the crude drug is
calculated from the mean of these 4 results.

31. The percentage of purity of the drug can be calculated using the following
equation.
N * W * 94000 * 100
% purity of drug = --------------------------------------------
S * M * P
Where, N = Number of starch grain in 25 fields.
W = Weight in mg of Lycopodium spore powder
S = Number of Lycopodium spores in the same 25 fields
M = Weight of sample in mg
P = No. of starch grains per mg present in the drug sample
(e.g. 1 mg ginger powder contains 2,86,000 starch grains)
94000 = Lycopodium spore count per mg

32. CAMERA LUCIDA

33. Camera lucida
• Camera lucida, (Latin: “light chamber”), optical instrument
patented in 1806 by William Hyde Wollaston to
facilitate accurate sketching of objects.
• Camera lucida is an optical device which when connected to
a microscope can help a person to draw the image of an
object in scale with the actual object by tracing on the
superimpose image.
• The principle is very simple, by looking into the prism from
just right angle, two images will enter the eye; one of the
object to be traced and other of the paper.
• The resulting effect is that your eye perceives illusion of
seeing objects in front of the instrument on the drawing
surface beneath.
• This is possible only after proper angle adjustment between
mirror/prism and drawing board.

34. It works on simple optical principle reflecting beam of light
through a prism and a plane mirror.
Thus it is a unique tool for the recording of fine details

35. Types of camera lucida
Prism type Mirror type

36. Parts of camera lucida
• There are three main parts of camera lucida –
1. Attachment ring
2. Prism
3. Mirror
• Attachment ring attaches camera lucida around the body
tube and prism over eyepiece.
• Prism and mirror reflects the image on paper.

37. PHYSICAL EVALUATION

38. • Evaluation of the drugs on the basis of important physical
properties or physical characteristics of the active constituent is
known as physical evaluation.
• Physical standards are constant for crude drugs and help in
evaluation of crude drugs.
• The characteristics studies involved in physical evaluation are
given in following table.

39. No. Class Method
1. Foreign material Moisture content
Foreign organic matter
Microbial contamination
Loss on drying
2. Ash values Total ash
Acid insoluble ash
Water soluble ash
Sulphated ash
3. Extractive values Water soluble extractives
Ethanol soluble extractives
Petroleum ether soluble extractives
Ether soluble extractives
Alcohol soluble extractives

40. 4. Physical constants Melting point
Boiling point
Optical rotation
Rf value
Solubility in different solvents
Bitterness value
Swelling index
Viscosity
Specific gravity
pH value
Density
5. Chromatographic Methods Thin layer chromatography (TLC)
High performance liquid chromatography (HPLC)
Gas liquid chromatography (GLC)
High performance thin layer chromatography
(HPTLC)
Column chromatography
Gel permeation chromatography( Gel filteration)
Affinity chromatography

41. 6. Spectroscopical
Methods
UV and visible spectroscopy
Infra-red spectroscopy
NMR spectroscopy
Mass spectroscopy
X-ray spectroscopy
Radio immune assay (RIA)
Fluorescence analysis

42. 1. FOREIGN MATERIAL :
• A) Moisture content :
 An excess water in medicinal plant material will lead to deterioration
through microbial growth or enzyme mediated hydrolysis in glycoside
containing plants.
 Therefore limits for the amount of water should be set for every plant
material.
 The moisture content of a drug should be minimized to prevent
decomposition of crude drugs either due to chemical change or
microbial contamination.
 Moisture content is determined by following methods:
i) Loss on Drying (dry in oven at 100-105 °C)
ii) Azeotropic (toluene distillation) distillation method
iii) Karl Fischer method

43. (a) Foreign organic matter:
 The parts of the organs other than the crude drugs are defined as
foreign organic matter.
 The maximum limit for the foreign organic matter is given in
monographs of crude drugs.
 If the limit exceeds, it indicates deterioration in quality of crude drugs.
(a) Microbial contamination:
 The drugs such as Acacia, Agar, Tragacanth, powdered Digitalis etc.
should be free of E. coli microorganisms.
 The crude drugs to be taken internally, should be free from bacteria
and mold contamination.
(a) Loss on drying:
 The loss in weight of crude drugs due to drying of water and volatile
oil.

44. 2. Ash values :
 The residue remaining after incineration of drug is the Ash
content of the drug, which simply represents inorganic salts,
naturally occurring in drug or adhering to it or deliberately
added to it, as form of adulteration.
 It varies in cases of many crude drugs and its study gives an idea
about the quality and purity of the drug during evaluation.
(a)Total Ash:
• The test is designed to measure the amount of material
remaining after ignition.
 Carbon and organic matter present in the drug is converted to
ash at the 450˚C or above.
 It contains carbonates, phosphates, silicates and silica.
 Many a time, the crude drugs are admixed with various mineral
substances like sand, soil, chalk powder or other drugs with
different inorganic contents.

45. b) Acid insoluble ash:
 Acid-insoluble ash is the residue obtained after
treating the total ash with dil. HCl.
 This determination measures the presence of
silica and excessive earthy material.
c) Water soluble ash :
• It is the calculated difference in weight
between the total ash and the residue
remaining after treatment of total ash with
water.
Incinerator

46. 3. EXTRACTIVE VALUES:
• The crude drugs have their biological activity due to chemical
constituents.
• These constituents are soluble in different solvents.
• It is the amount of active constituents extracted with solvents
from a given amount of medicinal plant material.
(a)Water soluble extractive:
 This method is applied to drugs which contain water soluble
active constituents of crude drugs, such as tannins, sugars, plant
acids, mucilage, glycosides etc.

47. (b) Ether soluble extractive:
 The types of ether soluble extractives values determined for
evaluation of crude drugs are volatile and non-volatile ether
extractives.
 The volatile ether-soluble extractive represents volatile oil
content of the drug.
 Non-volatile ether soluble extractives represent resin, fixed oils,
or coloring matter present in the drugs.
(c) Alcohol soluble extractive:
 Alcohol is an ideal solvent for extraction of various chemicals like
tannins, resins, etc.
 95% ethyl alcohol is used for determination of alcohol soluble
extractive.

48. 4. Physical constants
(a) Melting Point:
 Melting point is used to judge the purity of crude drug.
 In case of pure chemicals or phytochemicals, melting points are very
sharp and constant.
 The drugs from animals and plants origin contain the mixed
chemicals; they give certain range of melting point.
 The purity of crude drugs can be ascertained by determining their
melting points in the range.
(b) Boiling point:
• This parameter is applicable to all liquid phytochemicals like oils or few
alkaloids.
• Shift in boiling point range helps in determining purity of
phytochemicals.

49. (c) Refractive index:
 When a ray of light passes from one medium to another of
different density, the ray is bent from original path.
 The ratio of the velocity of light in air to its velocity in the
substance (drug) is termed as refractive index of the substance.
 Refractive index is constant for liquid depending upon purity and
it is considered for its standardization.
 Refractive index of a compound varies with light, temperature
and pressure.

50. (d) Optical rotation:
 Many volatile oils and other substances show the ability to
rotate the plane of the polarized light to right or left side and
likewise they are called dextrorotatory or levorotatory
respectively.
 This detection of optical rotation and its magnitude is an
important criteria for evaluation of drugs.
(e) Rf value:
 The active constituent present in the crude drugs when
subjected to thin layer chromatographic study, move on the TLC
plates according to their affinity with the solvent.
 The quality control employs TLC for assessing the quality and
purity of the drug.
 Rf value is the ratio of distance moved by the solute divided by
the distance moved by the solvent front.
 It varies from zero to one.

51. (f) Solubility in different solvents:
 The presences of adulteration in the drug could be identified by
solubility studies.
 e.g. Castor oil is soluble in 90% alcohol.
 Balsam of Peru is soluble in chloral hydrate solution.
 Asafoetida is soluble in carbon disulphide.
 Alkaloidal bases are soluble in chloroform, while alkaloidal salts are
soluble in polar solvents.
 Colophony is freely soluble in light petroleum.
(g) Bitterness value:
• Medicinal plant materials that have a strong bitter taste.
• Bitter substances can be determined chemically. However, since they
are mostly composed of two or more constituents with various degrees
of bitterness, it is first necessary to measure total bitterness by taste.
• The bitterness value is determined organoleptically by comparison with
a Quinine hydrochloride solution which acts as the standard.

52. (h) Swelling index:
• Many medicinal plant materials are of specific therapeutic or
pharmaceutical utility because of their swelling properties,
especially gums and those containing an appreciable amount of
mucilage, pectin or hemicellulose.
 Swelling index is defined as the volume in ml occupied by
swelling of 1 gm of a drug, under specified condition.
 The mixing of whole plant material with the swelling agent is
easy to achieve, but cut or pulverized material requires vigorous
shaking at specified intervals to ensure even distribution of the
material in the swelling agent.

53. (i) Viscosity:
 Viscosity of a liquid is constant at a given temperature.
 Viscous natural drugs like gums, mucilage or pectin like
compounds should be evaluated for its viscosity.
 Hence it can be used as a means of standardizing liquid drugs.
(j) Density and Specific gravity:
 All liquid phytochemicals have to be evaluated for its density and
specific gravity.
 This parameters is very essential for volatile oil standardization.
 specific gravity, is the ratio of the density of a substance to the
density of a given reference material.

54. CHROMATOGRAPHY
Greek word CHROMA means COLOR
And
GRAPHEIN means TO WRITE

55.  Chromatography is a separation technique for separating
components of a mixture. The different components of the
mixture travel through the stationary phase at different speeds,
causing them to separate from one another.
 Separation depends on the differential in affinities of the solute
between two immiscible phases.
 A stationary phase and mobile phase.
 The stationary phase is a fixed bed of large surface area which
may be porous of finely divided solid or liquid that has been
coated as a thin layer on inert support material.
 The mobile phase is moving phase over stationary phase.
 Mobile phase may be a pure liquid or a mixture of solutions or it
may be gas or mixture of gases.

56. TYPES OF CHROMATOGRAPHY
1. Paper chromatography
2. Thin layer chromatography (TLC)
3. High performance thin layer chromatography
(HPTLC)
4. High performance liquid chromatography (HPLC)
5. Gas chromatography
6. Gas liquid chromatography (GLC)
7. Column chromatography

57. 1. Paper Chromatography
• Paper Chromatography can be used to separate mixtures of
colored compounds.
• Mixtures that are suitable for separation by chromatography
include inks, dyes and amino acids.
• A spot of the mixture is placed near the bottom of a piece of
chromatography paper.
• The paper is then placed upright in a suitable solvent , such as
water, Methanol, Chloroform, etc.
• As the solvent soaks up the paper, it carries the mixtures with it.
• Different components of the mixture will move at different rates.
This separates the mixture out.

59. Rf value :
• Different chromatograms and the separated components of the
mixtures can be identified by calculating the retardation
factor (Rf).
• The Rf value is worked out by using this equation:
• Rf = distance traveled by the solute / distance traveled by the
solvent
• It varies from zero to one.
• The Rf value of a particular compound is always the same if the
chromatography has been carried out in the same way.
• This allows industry to use chromatography to identify
compounds in mixtures.
• Chromatography can also be done when the different substances
in the mixture are colorless.
• The chromatogram can be exposed to a locating agent (Spraying
reagent), which reacts with the invisible chemicals so that they
can be seen.

60. 2. Thin Layer Chromatography (TLC)
• Stationary phase in TLC is silica plate.
• The adsorbent such as silica gel G or C is coated 0.3 mm thickness
on clean TLC plates using spreader, the plates are heated at 105˚
for 30 minutes and used. Detecting agent is sprayed.
• TLC technique is useful in analysis of alkaloids, glycosides,
isoprenoids, lipids, sugars, derivatives and all phytoconstituents.
• The Rf values may vary depending upon purity of solvent,
substances, composition, impurities, adsorbent, polarity etc.
• This method is simple to operate, rapid and economical for
investigation.

61. 3. High performance thin layer chromatography
 HPTLC is very useful in qualitative and quantitative analysis as
quick and economical.
 Silica gel is used as adsorbent in HPTLC.
 Sampling is done by instrument (auto sampling)
 A sample of high concentration in small amount is used.
 Self-loading capillaries or linear development method is used.
 Specific detection is there in HPTLC, e.g. detection of spot
under UV light (different wavelength)
 HPTLC is used for analysis of glycoside, alkaloids, flavonoids.

63. 4. Gas liquid chromatography
 For separation of volatile substances by percolating a gas stream
over a stationary phase.
 Stationary phase : Liquid
 Mobile phase : Gas (Helium)
 The basis of separation of the sample in and out of the film of
liquid spread over an inert solid up to 450˚C with nitrogen or
helium gas.
 GLC is used for testing
i) starting materials or drug,
ii) quantification of drug in formulations and
iii) impurities or solvents in raw material or in drug.
 GLC examine volatile oils, plant acids, alkaloids of opium,
tobacco, conium and belladonna, glycosides, etc.

64. 5. Column chromatography
 It is liquid chromatography with liquid in mobile phase passes
over stationary phase packed in column.
 The column is either a glass or metallic one.
 Column chromatography can be done using gravity to move the
solvent, or using compressed gas to push the solvent through
the column.
 The adsorbents like starch, calcium carbonate, magnesia, lime,
silica gel, alumina, charcoal are used.
 It is used to isolate a single chemical compound from a
mixture.

66. 6. High performance liquid chromatography (HPLC)
• The separation principle of HPLC is based on the distribution of
the analyte (sample) between a mobile phase (eluent) and a
stationary phase (packing material of the column).
• Depending on the chemical structure of the analyte, the
molecules are retarded while passing the stationary phase.

67. SPECROPHOTOMETRY
1. UV-visible spectrophotometry
2. Mass spectrometry
3. Nuclear magnetic resonance spectroscopy
4. Infrared spectroscopy
5. Radio immune assay
6. Fluorescence analysis

68. 1. Ultraviolet – visible spectroscopy
 Ultra-violet (UV) and visible absorption techniques contain analytical
methods based on measurement of light absorption by substances in
the wavelength region from 180 to 780 nm.
 The Principle of UV-Visible Spectroscopy is based on the absorption of
ultraviolet light or visible light by chemical compounds, which results
in the production of distinct spectra
 The region from 180 to 390 nm is known as the UV region and from
390 to 780 nm. the visible region of the spectrum.
 Absorption in the UV visible region arises from electronic transitions
within the molecule.

70. 2. Infra red spectroscopy
• Infrared spectroscopy is a very powerful technique which uses
electromagnetic radiation in the infrared region for the determination
and identification of molecular structure as well as having various
quantitative applications within analytical chemistry.
• Wavelength range from 700 nm to 1 mm
• atoms can absorb energy from electromagnetic radiation; this
absorbed energy alters the state of the atoms within the molecule.
These changes are usually manifest in alterations to the frequency and
amplitude of molecular vibrations, which may be measured and plotted
to produce an infrared spectrum.
• Infrared spectroscopy is one of the most useful and widely used
methods to perform structural analysis, since it is helpful to identify
functional groups.
• IRS is used for the identification of drugs, polymorphic modifications,
excipients and raw materials, due to its sensitivity.

72. 3. Nuclear magnetic resonance (NMR) Spectroscopy
 NMR is the branch of spectroscopy dealing with the absorption of
radio frequency radiation by substances held in a magnetic field.
 Absorption results from interaction of radiation with magnetic
movement of nuclei in the sample at it occurs at different frequencies
for nuclei with chemically different environments within a molecule.
 NMR is important for elucidation of molecular structure, especially
the stereochemistry and configuration.
 The process reveals position of protons in a complex molecule.
 This is useful for determination of Hydrogen bonding (Intermolecular
and intramolecular) and Carbon bonding.
 It determinates impurities and minor components in mixtures as
comfort, speed and specificity of analysis.
 It is used to identify structural isomers

73. 4. Mass spectrometry
 In mass spectrometry, molecules in gaseous state under the pressure
between 10-7 to 10-5 mmHg are bombarded (attacked) with beam of
energetic electrons using tungsten or rhenium filament.
 The molecules are ionized and broken up in fragments, which can
further break up into smaller ions.
 All these ions are accelerated by an electric field, sorted out according
to their mass to charge ratio.
 The mass-to-charge ratio (symbols: m/z, m/e) of a cation is equal to
the mass of the cation divided by its charge. The mass of the
molecular ion is equal to the molecular weight of the compound. Thus,
the mass-to-charge ratio of the molecular ion is equal to the molecular
weight of the compound.
 The mass spectral data of compound indicates the molecular ion, peak
and other major and minor fragments which helps to establish the
structure of compound, by giving exact molecular weight, molecular
formula and an idea about its structural type.
 E.g m/e ratio of toluene is 91

74. 5. Fluorescence analysis
 The organic molecules absorb light over a specific range of wavelength
and many of them give out such radiations.
 Fluorescence is a process of re-emission of radiant energy absorbed
in the form of visible light.
 In this process light emitted is always of higher wavelength than that
absorbed.
 In fluorescence, absorption and emission of light takes place in very
short time (10-12 to 10-9 seconds.)
 If there is delay in the emission of light then the phenomenon is called
as phosphorescence. The delay period may range from fraction of
second to few days.
 Both the process called as luminescence.
 The drugs like belladonna leaf and root, wild cherry bark, gambier
catechu, aloes, jalap etc. show fluorescence in visible range.

75. CHEMICAL AND
BIOLOGICAL EVALUATION

76. CHEMICAL EVALUATION
• Qualitative chemical evaluation :
• Involves various chemical tests to identify different phytochemicals.
• Called as preliminary phytochemical screening.
• For example presence of alkaloids can be detected by dragondroff’s test,
Hager’s test, Wagner’s test, Mayer’s test
• Cardiac glycoside can be detected by Killer killani test
• Quinine can be identify by Thalleoquin test
• Quantitative chemical evaluation :
• Quantitative Assay, Values like Acid value, Saponification value for fats
and oil, Ester value, Aldehyde content, Acetyl value for volatile oil and
quantitative estimation of individual phytochemicals by using
chromatography, spectroscopy, etc. are parameters of quantitative
chemical evaluation.

77. No Chemical groups Chemical tests Result
1 Alakaloids Dragendorff’s test
Extract + Dragendorff’s
reagent
Yellow/ orange
precipitate
2 Glycosides Molisch’s test
Extract + α- naphthol + 1 ml
conc. Sulphuric acid
Violet ring at
junction of two
layers
3 Steroidal glycoside Lieberman Burchard Test
Extract + 2 drops of
concentrated sulphuric acid.
Green colour
4 Anthraquinones
glycoside
Borntrager test
Acid extract + benzene +
Ammonia
Pink ammoniacal
layer
5 Flavonoids Shinoda test
Extract + Mg turnings + 2
drops of conc. HCl
Pink colour
6 Tannins Ferric chloride test
Dilute extract + 2 ml ferric
chloride solution
Dark blue colour
7 Reducing sugar Fehling solution test
Extract + Fehling solution A
and B- boiling on water bath
Pink red
precipitate
8 Mucilage Extract + 1 ml of rhuthenium
red solution
Pink colour

78. BIOLOGICAL EVALUATION
 When the estimation of potency of crude drug or its
preparation is done by means of its effect on living organisms
like bacteria, fungal growth, or animal tissue or entire animal. It
is known as bioassay.
 Such activity is represented in units known as International unit
(I.U.).
 The specific biological activity contained in each I.U. of the few
drugs is given below:
o Digitalis: 1 I.U. is contained in 76 mg of standard preparation.
o Vitamin A: 1 I.U. is present in 0.344 micrograms of standard
preparations.
o Vitamin D: 1 I.U. is contained in 0.025 micrograms of
standard preparations.

79.  Biological assay methods are of 3 types:
(i) toxic
(ii) symptomatic
(iii) tissue methods.
 In toxic and symtpmatic tests, the animals are used and in tissue
method, the effect of a drug is observed on isolated organ or tissue.
 Biological testing of herbal drugs:
 In standardization/evaluation of herbal drugs, assessment of biological
efficacy is found to be most assuming method.
(a) Hepatoprotective activity:
 Male/female albino rats are used.
 Inducing chemical : Allyl alcohol, cause liver necrosis, Paracetamol
induced liver damage, Carbon Tetrachloride
 Standard drug : Silymarin

80. 2. Anti-diabetic activity :
• Animal – Mice or Rat
• Inducing chemical – Alloxan, Streptozotocin
• Standard drug – Glipizide, Glibenclamide
3. Anti-ulcer activity :
• Animal – Rats
• Inducing chemical – Indomethacin, Immobilization stress
• Standard drug – Omeprazole
4. Anti-inflammatory activity :
 The drugs from plant origin cause anti-inflammatory effects and used in
conditions like rheumatoid arthritis, gout, etc.
 The principle underlying the testing of anti-inflammatory activity is the
reduction of local oedema induced in rat or Mice paw by injecting irritant,
inflammatory substances like Formaldehyde, Carrageenan, Egg Albumin,
Dextran, Ultraviolet Light
• Standard drug – Diclofenac, Indomethacin,