INDEX
Sr. Experiment name Date Page no. Sign
no.
1 To prepare and submit herbarium 13/09/2022
2 13/09/2022
3 To study various parts of compound 20/09/2022
microscope
4 To determine the stomatal number and 20/09/2022
stomatal index of vasaca stramonium
leaf.
5 Introduction to Extraction Procedures. 17/10/2022
6 To extract caffeine from Tea. 17/10/2022
7 To prepare herbal colours 18/10/2022
Experiment no. 01
Aim: To prepare and submit herbarium.
Reference ; Dr. Sourabh kosey, A practical book of Practice school by Nirali
prakashan first edition September 2021 page no. 65-66
Theory
An Herbarium is defined as a collection of plants that usually have been dried, pressed,
preserved on sheets, and arranged according to any accepted system of classification for future
reference and study. In fact, it is a great filleting system for information about plants, both
primary in the form of actual specimens of the plants, and secondary in the form of published
information, pictures and recorded notes.
Methods of preparation of herbarium specimens:
The preparation of a herbarium involves:
(i) Field visits
(ii) Collection of specimens
(iii) Drying
(iv) Mounting on a herbarium sheet
(v) Preservation
(vi) Labelling and
(vii) Proper storage
(a) Field visits and specimen collection: A complete specimen possesses all parts
including root system, flowers and fruits. Therefore, regular field visits are necessary
to obtain information at every stage ot growth and reproduction of a plant species. In
the fields, the tools required are mainly trowel (digger) for digging roots, scissors and
knife for cutting twigs, a stick with a hook for collection of parts of tall trees, a field
note book, polythene bag, old newspaper and magazines.
To avoid damage during transportation and preservation at least 5-G specimens of a
plant should by collected. The collected specimens are transported in a vasculum
(specimen box) to prevent willing, livery collected specimen must be tagged with a
field number and necessary information should be recorded in a field note book.
(b) Pressing and drying: The specimens are spread out between the folds of old
newspapers or blotting sheets avoiding overlapping of parts. The larger specimen
may fold in 'N' or 'W shapes. the blotting sheets with plant specimen should be
placed in the plant press tour drying. After 24 to 48 hours the press is opened.
(c) Mounting: The dried specimens are mounted on herbarium sheets of standard size (41 x
29 cm). Mounting is done with die help or glue, adhesive or cello-tape. The bulky
plant parts like dry fruits seeds, cones etc. are dried without pressing and are put in
small envelops called fragment packets. Succulent plants are not mounted on
herbarium sheets but are collected in 47% formalin or FAA (Formalin Acetic Alcohol).
(d) Preservation: The mounted specimens are sprayed with fungicides like 2% of
mercuric chloride. solution
(e) Labelling: A label is pasted or printed on the lower right-hand corner. The label
should indicate the information about the locality, altitude, habit, date and lime of
collection, name of collector, common name, complete scientific name etc.
(f) Storage: Properly dried, pressed and identified plant specimens are placed in thin
paper folds (specimen covers) which are kept together in thicker paper folders genus overs),
and finally they are incorporated into the herbarium cupboards in their proper position
according to a well-known system of classification. In India Bentham and Hooker's system of
classification is used for' his purpose. Type specimens are generally stored in
separate and safe places.
Reports:
Experiment no. 02
Aim: Monograph of
Experiment no. 03
Aim: To study various parts of compound microscope.
Reference : Dr. Sourabh kosey, A practical book of " Practice school" by Nirali
prakashan , first edition September 2021, page no. 68-69.
Theory:
The microscope is a biologist's basic tool. It has been developed to help explore the
world of living things too small to be seen with the naked eye. Early microscopes had only one
lens and were difficult to use. The biggest problem was magnification. The more powerful the
lens needed for greater magnification meant the closer the viewer's eye had to be to the lens. At
very high magnification, the lens almost touched the eye. The microscope early user had to be
very steady. A major advance in microscopes came with the invention of the compound
microscope. It has two se*s of lenses, which magnify objects much greater than a single lens
Structure of the Microscope:
The compound microscope has four basic parts: the lens system, the focusing
system, the stage, and the lighting system.
The Lens System:
One of the two sets of lenses is the objective lenses. They work similarly to the lens of the
early, simple microscope. The objective lenses make the initial or primary magnification.
They are located in the nosepiece of the microscope. Inscribed on each objective is the
magnification or power of that lens. This tells the number of times the lens magnifies the
image. For example, if you are looking at a strand of hair with a 4x (four-power) lens, the hair
will appear four times its actual size. Your microscope probably has at least two objective
lenses. Some microscopes have as many as four objectives. Rotate the lenses in the
nosepiece until they click into position. The objective lens in use is always.
The Stage:
A specimen to be viewed through the microscope is mounted on a glass slide and covered with
a cover slip. The slide rests on the stage, the flat surface beneath the body tube. Stage clips hold
the slide in place. Also, they help in making slight adjustments in the slide's position by holding
the slide steady. The stage should always be kept in a horizontal position. If you tilt the stage, the
specimen will slip to the bottom edge of the slide. Even with commercially prepared slides, the
stage should be kept horizontal. A commercial sloe can be ruined as the cover slip slowly slips
downward on a tilted stage. Both the slide anu the stage is extremely smooth. Water between
them acts like glue and causes the slide to stick to the stage. If water gets on the stage, STOP
and dry both the stage and the bottom of the slide with a paper towel before proceeding.
The lighting System:
For you to see the specimen, light must pass through it and the lenses to your eye. The lighting
system is located under the stage of the microscope. There are three different types of lighting
systems. The simplest system uses a concave mirror to focus a beam of light on the slide. Tilt
the curved surface of the mirror to face a light source: room lights, windows, or a desk lamp.
Another lighting system uses a lens under the stage to focus the light. If there is a substage lens
and a mirror on your microscope, use the flat side of the mirror to reflect light through this lens. A
third lighting system uses a sub-stage light instead of a mirror. If your microscope has
The Focusing System:
In order to bring the image of the specimen into proper focus, it is necessary to change
the distance between the slide and the objective lens. This can be done in one of two
wavs. depending upon the microscope you are using. Either the lenses can be moved or
the stage upon which the slide rests can be moved. Two knobs control the focus. The
coarse adjustment knob is for coarse focusing, and the fine adjustment knob is for fine
focusing. locate these on your microscope. Turn the coarse adjustment knob.
Experiment no. 04
Aim: To determine the stomatal number and stomatal index of vasaca stramonium leaf.
Requirements:
Compound microscope, stage micrometre, camera Lucida, drawing paper, chloral
hydrate solution and glycerine water.
Principle/ theory:
Stomatal number is defined as the average number of stomata present per square mm
of epidermis of the leaf. Stomatal index is the percentage which the numbers of stomata
form to the total number of epidermal cells, each stoma being counted as one cell.
Procedure:
• Clear the fragments of leaf from the middle of lamina by boiling with Chloral
Hydrate Solution or Alternatively with chlorinated Soda. Peel out upper
epidermis and also the lower one separately by means of forceps.
• Arrange a camera Lucida and drawing paper for marking the drawings.
• Mount the lower and upper epidermis separately in glycerine water.
• Draw a square of 1 mm with the use of a stage micrometeor and camera
Lucida on a drawing paper.
• Replace the stage micrometre by the cleared leaf preparation, focus under the
same magnification and trace the epidermal cells and stomata by looking through
the microscope when a superimposed image of the leaf is seen at the same time.
• Count the number of epidermal cells and stomata (the two guard cells and
ostiole being considered as one unit) within the square, a cell being counted if
at least half of its area lies within the square, provided two adjacent sides are
considered for purpose of calculation.
• Record the result for each of the ten fields and calculate the stomatal number
i.e., number of stomata per sq. mm. or leaf preparation,
• calculate the stomatal Index using the formula:
Stomatal Index S.I. = S/E + Sx 100 g.
• Determine the values for each surface where the leaf bears stomata on both surfaces.
Result:
Stomatal Index S.I. = S/E + S x 100 g.
Experiment no. 05
Aim: Introduction to Extraction Procedures.
Reference : Dr. Sourabh kosey, A practical book of Practice school by Nirali
prakashan first edition September 2021 page no. 70-75.
Theory:
Extraction is defined as the process of isolation of therapeutically active soluble chemical
constituents (secondary metabolites) from the crude drugs by treatment with selective
solvent. Solvent used for extraction can be distilled water or organic solvent and it is
called menstruum. Extraction is controlled by unit operation known as mass transfer.
Mass transfer involves the transfer of mass of soluble material from solid to fluid. When
crude dug is placed in contact with solvent, the solvent start penetrating inside the cells
and constituents. the dissolved constituents diffuse through the cell subsequently forms
the solution of the constituents within the cells containing active chemical constituents.
The dissolved constituents diffused through the cell wall and boundary layer into the
solvent used for the extraction. Extraction depends upon various factors such as particle
size of the crude drugs, method of extraction, polarity of solvent and temperature etc
extraction process may be solid-liquid extraction or liquid-liquid extraction.
Methods of Extraction:
extraction methods can be categorized according to the required yield.
(a) Small Scale Extraction Process: Maceration, Percolation, Infusion,
Decoction, Digestion
(b) Continuous Extraction Procedure: Soxhlet extraction
(c) Large Scale Extraction Process: Supercritical fluid extraction, counter-
current Extraction
Maceration:
Maceration is process of extraction of active chemical constituents from crude drugs. It s
carried out by immersing coarsely powdered crude drug in bulk of solvent placed in
stoppered glass container known as macerator. The solid drug material and solvent (approx.
750 ml) is allowed to stand for at least 3-7 days in a warm place with frequent shaking. The
mixture is then filtered until most of the liquid is drained off after washing. The marc (the
Gamp solid material) is pressed. The filtrate and the washing are combined to produce final
volume of the solution. Maceration can be modified to multiple-stage extraction to increase
yield of active chemical constituents in the extracts. Maceration can be of three types:
1 Simple maceration
2. Maceration with adjustment
3. Multiple maceration
a) Double maceration
b) Triple maceration
Applications:
Maceration can be carried out for tincture made from organized drugs and unorganized drugs.
Aqueous extract of the crude drugs is made by maceration.
Infusion: Infusion is prepared by infusing the drug with 8 parts of water for 15-20 min and
then straining with muslin cloth. Infusion pots are the simplest form of apparatus used for
preparing infusion. Infusion pots consist of loose perforated shelves on which drug is placed
and allowed to immerse in water for extraction. the active chemical gets dissolved in water
and the liquid is filtrate off after occasional stirring. The marc is not pressed and the final
volume is not adjusted with solvents. However hot infusion is prepared by infusing the drug
with hot water since it has greater solvent action. Infusion should be stored in well-closed
container protected from the air and light. It should be used within 12 hours after its
preparation as it can be spoiled by microbial contaminants. Infusion is of two types.
1 Dilute Infusions (Fresh infusion)
2 Concentrated Infusions
Applications: It is used to make dilute solutions of readily soluble chemical
constituents of crude drugs.
Digestion:
Digestion is heating the drug with specified solvent at particular pressure .This increases
the penetration power of menstruum in order to carry out complete efficient atraction.
Temperature should be increased in a controlled manner so that it could not troy the
chemical constituents. Extraction is carried out in apparatus made up of netal known as
Digestor. Drug along with solvent is placed in the body of the digestion or for specified
period of time under controlled temperature and pressure conditions. Lid is closed with
the help of bolts and nuts. To carry out the extraction the drug is treated with menstruum
for a definite period of time under specified conditions
Application: Digestion process is used in the extraction of enzymes and proteins
constituents from crude drugs
Percolation: It involves downward displacement of saturated solution formed during
extraction of active chemical constituents from crude drug by the slow passage of
menstruum through the column of the drug. The extracted solution is called percolate and
apparatus used for extraction is called percolator. After collecting the required volume of
extract solution or when the drug is completely exhausted, marc is pressed. The expressed
liquid is mixed with the percolate. Sufficient quantity of menstruum is added to produce the
required volume. Percolation involves three stages:
1. Imbibition of crude drug
2. Maceration of crude drug
3. Percolation of crude drug
Procedure:
Coarse or fine powdered drug is packed uniformly in the percolator. Before packing the
drug is allowed to imbibe in presence of solvent. It is passed through specified sieve to
break any lump or masses and then packed in percolator. Imbibition’s prevents blockage
of percolator and also allows the entrapped air to escape. The drug should be uniformly
th
packed upto 2/3 or 3/4 of the percolator in order to facilitate free flow of the menstruum
through the drug. Quantity of menstruum to be used depends upon the swelling power of
ua
the drun. Sufficient q ntity is added to saturate the drug material. Outlet of percolator is
closed when the solvent start coming out from the percolator. The dug is allowed to
remain in contact with the menstruum for approx. 24 hours. During the period of
maceration, the menstruum penetrates into the tissue of the drug and extracts the active
chemical constituents. After specified period of time outlet is opened and percolate is
collected. Percolation is continued till the drug gets exhausted. Marc is collected and
pressed. The expressed liquid is mixed with the percolate. Final volume is made by
adding more menstruum. The liquid extract is filtered to remove foreign particle if any.
Types:
Percolation is of the following types:
1. Simple percolation process
2. Percolation processes for concentrated preparation
(a) Reserve Percolation Process
(b) Modified percolation process
Application :
Simple percolation process used for preparation of tinctures of phytodrugs. It is used
to make extract of crude drugs when quantity of solvent is limited for use Soxhlet
extraction or Continuous Hot Percolation Process: Soxhlet extraction is the process
of continuous extraction in which the same solvent be circulated through the
extractor for several times. This process involves extraction followed by the
evaporation of the solvent. The solvent vaporize into the condenser a the condensed
solvent fall back on the crude powdered drug for continuous extraction.
Apparatus: Soxhlet apparatus consist of body of extractor attached with a side tube
and siphon see The lower side of extractor is attached to distillation flask and mouth
of the extractor is d to condenser by the adjusting joints.
Procedure:
Crude drug powder is packed on thimble of filter paper or fine muslin in the soxhlet exaractor.
Extraction assembly is assembled by fixing the distillation flask and the condenst initially settling
of powder solvent is allowed to siphon once before the heating. Fresh Porcelain pieces are
added to flask to avoid bumping of the solvent. On heating vapors pass trough the side tube and
the condensed liquid gradually increase the level of solvent in the extractor and siphon tube. As
the level of solvent rise in the siphoning tube the contents of the extraction chamber are
transferred into the flask. The cycle of solvent evaporation wing of condensed solvent in the
extractor and siphoning continues many times Still complete extraction of the crude drug material
occurs. Similar methodology can be adopted by large scale extraction purpose.
Supercritical fluid extraction:
The supercritical fluid extraction ”is comparatively newly method of extraction of crude Bugs In
this case extraction is carried out with supercritical fluids. These fluids are actually gases which
behave like free-flowing liquids at critical point of temperature and pressure. Such supercritical
fluids have very high penetration power and extraction efficiency. The mature to be fractionated
is passed in the extraction column along the length of which heater is located. CO, (supercritical
fluid) is purged through the column. Once the extraction pressurized drug material gets saturated
in the supercritical fluid which moves along with the length of the column. The operating
conditions ie temperature and pressure is selected and operation of super critical fluid extraction
is controlled by Programmer Logical Controlled device.
Application:
The process is successfully used in isolation of caffeine from coffee beans, extraction of
Pyrethrums’ and for the production of terpene less oil. It is used selectively in the
extraction of acorone from calarnus matricin and bisabol from chamomile flowers.
Counter Current Extraction:
Crude drug material is first pulverized before extraction. Fine slurry of the material to
be extracted is allowed to move in one direction with cylindrical extractor. Solvent is
allowed to move in opposite direction under specific pressure and temperature. Flow
rates are optimized to carry out the efficient extraction. Finally, the concentrated is
collected from one and the marc is pressed towards the other end.
Application:
It Is used for extraction of thermo liable chemical constituents moreover the
extraction procedure is more efficient and effective than continuous hot extraction.
Experiment no.06
Aim : To extract caffeine from Tea.
Reference : Dr. Sourabh kosey, A practical book of Practice school by Nirali
prakashan first edition September 2021 page no. 76-77.
Requirements: Tea Leaves, Distilled water, Lead acetate solution, dil.sulphuric acid,
chloroform, Water bath, separating funnel, filter paper, beaker, funnel glass rod,
tripod stand etc.
Theory:
Tea is obtained from the prepared leaves and leaf buds of Theo sinensis belonging
to family Theaceae. Caffeine occurs as white powder without any odor but possess
bitter tas It is a weak base and freely soluble in water is sharply increased in the
presence o benzoates, bromides, citric acid and salicylates
Procedure:
1) Dissolution of caffeine in water:
i) Weigh about 15 gm of tea leaves and place them in a beaker of 50-100 ml and ad
30 mL of distilled water to the beaker.
ii)Boil the water containing tea bags on boiling water for 15-20 min while stirring
Occasionally. iii)After the boiling period is over, remove the beaker from heat and
allow cooling for 15 min. on ice water.
iv)After the sol, has cooled, squeeze the tea bag to remove all the liquid and
the bag. v)Using vacuum filtration, filter the sol. Through regular filter paper.
2)Removal of Tannins:
i)Transfer the obtained solution from the above step to 500 mL beaker and ad sufficient
amount of 10% lead acetate solution to precipitate tannin and other impurities.
ii) Filter the solution, remove precipitates and to filtrate add 10% sulfuric acid t
iii) Remove excess lead acetate as precipitates of lead sulfate.
iv) To the filtrate add 10% sodium hydroxide to basify the solution and again her the Solution.
3)Transfer of caffeine from water to chloroform :
i) Transfer the solution obtained from above step to 500 mL separating funnel
ii) Add 100 ml of CHCI3 and shake the separating funnel in a proper manner .
iii) Allow the Chloroform to settle to the bottom and carefully collect the CHCL3 layer
in the flask or beaker.
iv)Dispose off the aqueous Layer
v) Filter the CHCI, layer through reverse-phase after filter paper using Filtration.
vi) This will allow the CHCI, to filter through but will trap on of water and reside
Transfer the solution to a tare china dish.
4) Crystallization of caffeine
i)Using a hot water bath in a fume hood and place the CHCL3 solution over the boiling water.
ii) Evaporate the solution down to about 20 ml and remove from the heat.
iii) weigh the china dish containing caffeine and calculate the % yield
Calculation :
Practical yield = weight pf product/weight of the sample × 100
Result:
Percentage yield of caffeine out to be ………….
Melting point = 50 -53 0C .
Experiment No: 07
Aim: To prepare herbal colours.
Reference : Dr. Sourabh kosey, A practical book of Practice school by Nirali
prakashan first edition September 2021 page no. 78
Theory/ Procedure:
1. Red : Dry hibiscus flowers until they are crisp and grind them into a fine powder. You can
also use red sandalwood for this one. You can add rice flour in equal quantities to
increase the volume of powder. For wet colours, boil peels of pomogranate in water.
2. Yellow: You can blend Turmeric powder with the ratio of 1:2 to make dry gulal
alternatively you may also use yellow coloured flowers, such as marigold or yellow
chrysanthemums can be crushed and combined in water as well for wet colours.
3. Green: To obtained a lovely green coloured gulal, you can use henna or mehendi
powder. In order To get a liquid paste, you can either mix the henna powder in
water or oil and mix with any green leafy vegetables such as Spinach.
4. Magenta : Soaked Sliced beetroots in water, boil the mixture and leave it
overnight. If you want a more pinkish shade, just dilute the concotion a little
more . Or you may also use red onions for this one.
5. Blue: Powdered blue hibiscus flowered petals and rice flour can be used to
obtained blue coloured gulal. For wet colours you can use dried and crushed
jacaranda flowers mixed with water.
6. Herbal Gulal: Take 100 gm arrow root powder , 100 gm haldi , 50 gm
marigold flowers, 20 gm orange peel powder ( finely powdered) and 20 drops
essential oil of lemon or sandalwood. Put all the Ingredients into the large
plastic mixing bowl or tub and hand mix , rubbing together gently. You will see
a beautiful yellow coloured, safe and natural gulal being made.
7. Dry powders with food colours: Take rice flour add proportionate food
colour and mix it with two teaspoons of water to make a thick paste. Leave it
to dry and then blend it in a grinder to mix and use it as a powdered colour.
8. Saffron tinge : For powdered, take besan and mix it with haldi with little water
and leave it in the sun to dry. For water colours soak henna leaves in water
overnight and use the water to play in holi. Also. The henna paste is ready.
Report :
