Komar university of science and technology
College of medicine
Department of pharmacy
Supervisor:Dr. V Gnanavel
-Course name
Pharmacognosy Lab report
-Prepared By:
1-Rakan Niemat ID:F210533
Section>= 1
May 22, 2022
Pharmacognosy
Lab Report

Experiment 1: Isolation of sennosides from Senna angustifolia leaves by maceration
Aim: To isolate and detect the active constituent, sennoside present in senna.
Sennoside is present in the dried compound leaflets of Cassia angustifolia belonging to the
family Leguminosae. The plant is a small shrub having 3-7 pairs of leaflets. The drug should be
protected from light during its storage. The major chemical constituent is sennoside-A and
sennoside-B which are anthraquinone glycosides. Both show stereoisomerism. Borntrager’s test
confirms the presence of anthraquinone. Sennosides are used to treat constipation. They may also
be used to clean out the intestines before a bowel examination/surgery. Sennosides are known as
stimulant laxatives. They work by keeping water in the intestines, which causes movement of the
intestines. It is used as purgative, stimulant laxatives.

Requirements
Apparatus: Extraction unit, Separating funnel, TLC plate, Evaporation unit, Funnel, Test
tube, Beaker, Measuring cylinder
Chemicals: Tea powder, ethanol, n-Hexane, 2N HCl, CHCl3, and acetone.
Procedure
1. Grind 50 g of dried senna leaves to a coarse powder in a mortar.
2. Macerate the powder in 100 mL of 70% ethanol with shaking at 100 rpm for 12 hours in
a glass reagent bottle.
3. Filter (thrice) the ethanolic extract with Whatman No.1 filter paper using
Buchner funnel (vacuum).
4. Concentrate the crude ethanolic extract in a rotary evaporator under a vacuum at 40° C
until thick extract is obtained.
5. Recover the condensed extract with enough warm deionized distilled water (20 mL).
6. Partition the recovered crude extract with equal volumes of n-Hexane (repeat the
fractionation procedure three times) in a separatory funnel.
7. Collect the aqueous phase and condense it under vacuum and reduced temperature in a
rotary evaporator.
8. Treat the condensed aqueous fraction with a sufficient quantity of 1-2N HCl until the
formation of ppt.
9. Filter the aqueous phase and recover with 10 mL CHCl3.

10. If the ppt. did not appear, then partition the aqueous phase with 20 mL CHCl3 (thrice),
use the organic phase, and evaporated under vacuum at reduced temperature (25° C) until
dryness.
11. Calculate the percentage of yield.
Detection of sennoside
Borntrager’s test: Different organic solvent like benzene, ether, chloroform is added to the
sample and shaken. The organic layer is separated and ammonia solution is added to it. The pink
or red color is produced by confirming the ammoniacal layer which shows the presence of
anthraquinone.
TLC of sennoside: The solvent system is ethyl acetate:methanol: water having a ratio of
100:16.5:13.5. The obtained purified sample is spotted in the plate of silica gel and developed in
ethyl acetate:methanol: water. When the dried plates of silica gel are sprayed with HNO3 (25%),
red-colored spots will be seen. After drying with alcoholic KOH red color is converted into
yellow color.
Discussion
Laxatives are one of the most commonly utilized pharmacological classes in the treatment of
digestive system functioning problems. Medicines that are commonly utilized in medical practice
The most common source of plant raw materials containing anthracene derivatives is Cassia
(Senna) - Senna leaves (Cassia acutifolia Del.) or Alexandrian Senna (Senna alexandrina Mill.) and
Cassia Angustifolia Vahl.)
S. alata Leaves Extraction Method Determination Due to the leaves of S. alata
Because S. alata contains both anthraquinone aglycones that are soluble in ethanol and glycosides
that are soluble in water, the solvent employed to extract S. alata leaf samples was water.
Maceration is an extraction method that is performed at room temperature. It entails immersing a
plant in a liquid (water, oil, alcohol, etc.) inside an airtight container for a varied amount of time
depending on the plant material and liquid utilized. Depending on the intended outcome, the plant
material might be utilized fresh or dried. S. alata powdered leaves were macerated in ethanol. The
extraction was repeated until the maceration extracts were exhausted (as determined by
Borntrager's reaction), then the maceration extracts were mixed, filtered, and evaporated to dryness
over a boiling water bath to obtain a maceration crude extract. Anthraquinones Identification
Anthraquinone aglycones in the extract were detected using Borntrager's reaction. The sample was
treated with 2M hydrochloric acid before being boiled in a hot water bath for 15 minutes, cooled,

and filtered. During this experiment, we had to grind 50 g of dried senna in a morter to a very fine
powder, then place it in a volumetric flask and add the ethanol. We filtered it using a funnel after
12 hours (Buchner funnel). The crude ethanol extract was then concentrated and evaporated at 40
degrees Celsius until it became a thick extract. Then it was refilled with water (approximately
20ml), which should be purified water. The recovered extract was then partitioned with an
equivalent amount of n-hexane. We repeated this process until we reached the aqueous phase, at
which point we collected it. We condensed it with Vaccum, chilled it, and then made ppt with 1-2
N HCL. The solution was then filtered and recovered with 10 mL of CHCl3. Finally, we would be
able to employ yield as medication by quantifying it. a liquid (water, oil, alcohol, etc.) in an airtight
container for a varying amount of time depending on the plant material and liquid used Depending
on the intended outcome, the plant material might be utilized fresh or dried. S. alata powdered
leaves were macerated in ethanol. The extraction was repeated until the maceration extracts were
exhausted (as determined by Borntrager's reaction), then the maceration extracts were mixed,
filtered, and evaporated to dryness over a boiling water bath to obtain a maceration crude extract.
Anthraquinones Identification Anthraquinone aglycones in the extract were detected using
Borntrager's reaction. The sample was treated with 2M hydrochloric acid before being boiled in a
hot water bath for 15 minutes, cooled, and filtered. During this experiment, we had to grind 50 g of
dried senna in a morter to a very fine powder, then place it in a volumetric flask and add the
ethanol. We filtered it using a funnel after 12 hours (Buchner funnel). The crude ethanol extract
was then concentrated and evaporated at 40 degrees Celsius until it became a thick extract. Then it
was refilled with water (approximately 20ml), which should be purified water. The retrieved extract
was then partitioned with an equal volume. containing n-hexane We repeated this process until we
reached the aqueous phase, at which point we collected it.
We condensed it with Vaccum, chilled it, and then made ppt with 1-2 N HCL. The solution was
then filtered and recovered with 10 mL of CHCl3. Finally, we would be able to employ yield as
medication by quantifying it.
Experiment 2: Isolation of Caffeine from tea dust.
Aim: To isolate and detect the active constituent, caffeine present in tea dust.
Caffeine is a purine base (1, 3, 7 – trimethyl xanthine). This is mostly produced from the tea
leaves and leaf buds synthetically. As green tea it is available from China and Japan and as black

tea from Sri Lanka and India. Leaves are green in colour, apex is blunt. Tea leaves are a rich
source of caffeine which is a weak base. Caffeine is present 1-4% in tea leaves while 1-2% in
coffee seed. Caffeine is a naturally occurring alkaloid produced by tea and coffee shrubs. It is a
CNS stimulant that is believed to act by serving as an antagonist of adenosine receptors on
neurons. Caffeine is soluble in hot water and is extracted from coffee grounds or tea leaves when
these products are brewed. While caffeine is water soluble, it is much more soluble in the organic
solvent methylene chloride (CH2Cl2). Methylene chloride is immiscible with water and when
mixed separates from water to form a two-layer mixture. Because methylene chloride is denser
than water it usually comprises the lower layer in the two-part mixture. By mixing brewed tea
with methylene chloride, the caffeine can be extracted into the organic layer. Since the organic

layer is immiscible with water, it can be removed after it separates from the water, and the
solvent evaporates to give nearly pure caffeine.
Requirements:
Apparatus: Extraction unit, Separating funnel, TLC plate, Evaporation unit, Funnel, Test
tube, Beaker, Measuring cylinder
Chemicals: Tea powder, calcium carbonate, Methylene chloride, magnesium sulphate, acetone
and petroleum ether.
Procedure:
Place 15 g of tea leaves, 5 g of calcium carbonate powder and 200 mL of water into a 600 mL
beaker. Boil the solution on a hot plate for 20 minutes with occasional stirring. Cool the solution
but, while it is still warm, vacuum filter through a Buchner funnel using a fast filter paper, if
available. Normally, hot solutions are not vacuum filtered. Rinse the leaves with 50 mL of water.
Carefully press out as much filtrate as possible since the caffeine is in the aqueous layer. Rinse
again with 50 mL of water. Cool the solution to room temperature and pour it into a 500 mL
separatory funnel. Extract with 35 mL of methylene chloride. In a departure from normal
procedure, it will be necessary to vigorously shake the separatory funnel in order to extract the
caffeine. First, relieve the pressure build up as soon as you mix the two liquids. Then shake
vigorously for 10 seconds and relieve pressure, repeat the shaking two more times. An emulsion
will probably form. To break the emulsion formed in the methylene chloride layer, slowly drain
the methylene chloride layer through a small amount of anhydrous magnesium sulphate in a
powder funnel with a loose cotton plug (a tight plug will prevent drainage). Extract the aqueous
solution once again with 35 mL of methylene chloride, repeating the steps above to collect the
lower layer. Combine the methylene chloride extracts and, if necessary, dry further with
additional anhydrous magnesium sulphate. The methylene chloride solution will be stripped on a
roto-evaporator. Tare weighs a 100-mL rb flask and transfers the dried methylene chloride

solution to it. Be certain that there is no magnesium sulphate in the solution. Stripping this
solution to dryness will take less than 5 minutes. You will be left with a small amount of residue
with a greenish tinge. Obtain the weight of crude caffeine by difference. Add 5-8 mL of hot
acetone to dissolve the crude caffeine and transfer the solution to a 50 mL Erlenmeyer flask for
recrystallization. Add a few drops of petroleum ether until you reach the cloud point (caffeine is
less soluble in this mixed solvent and is just beginning to precipitate) and then cool the solution.
If you do not get a precipitate, you may have used too much acetone, carefully boil off the excess
on a steam bath using a boiling stick for ebullition. Suction filters the caffeine using a small
Hirsch funnel and petroleum ether as a transfer/rinse solvent. A second crop of caffeine may
form in the filtrate as the solvent evaporates. This second crop can also be collected by vacuum
filtration but keep it separate from the first crop. After air drying, weigh each crop and record
your % caffeine recovered from tea. Collect the crystals by vacuum filtration, air dry, weigh,
record the yield, and take a melting point (lit mp 137 °C).
Discussion:
Boiling water is used to remove tea leaves (coarse powder form). The aqueous extract is filtered
under those heated conditions. The heated extract sample is treated with lead acetate to precipitate
the tannin component. Excess lead acetate in the sample is precipitated using dilute H2SO4. To
eliminate the coloring matter, the filtered sample is cooked with charcoal and then filtered again.
The filtered decolorized sample is extracted once again with chloroform. After evaporation, the
combined chloroform extract yields caffeine as a white substance, which may be re-crystallized
with alcohol.
The coarsely powdered tea leaves are extracted in a soxhlet extractor using ethanol. Caffeine has
been adsorbed on MgO in the extracted sample. Following a 10% treatment Caffeine is disrobed
using H2SO4. Finally, the sample is removed and re-crystallized using chloroform2.

Detection of caffeine
Murexide test: Caffeine are treated with a few crystals of HNO3 (3-4 drops) in a porcelain dish
and subjected to evaporation for drying. 2 drops of NaOH solution are added to the residue,
which gives a purple colour.
TLC of caffeine: 1 mg of caffeine is dissolved in 1 ml of methanol or chloroform. Using the
solvent (acetate:methanol:acetic acid, 80:10:10) the sample is eluted and spotted on the TLC
plate. By exposing to vapour of iodine, the dried plates are visualized at an Rf value of 0.40-0.42.
Conclusion: The active constituent caffeine was isolated and detected from the leaves of tea. It
can be used as CNS stimulant, specific analgesic in migraine.
Calculation of percentage yield:
REPORT:
1. % Yield of Caffeine =
2. Melting point of Caffeine =227-228 °C

Experiment 3: EXTRACTION OF STARCH FROM POTATO
Aim:
The aim is to extract the starch from a sample of potatoes.
Principle:
Starch is a carbohydrate that can be found in plants. Starch appears in the form of granules under
the microscope. Individual starch granules have this characteristic. Depending on the source
from which they were isolated, they vary in size. Insoluble in water, starch sinks to the bottom
quickly and can be collected by decanting the supernatant.
Materials Required:
Potato, Muslin Cloth, Watch Glass, Mortar and Pestle, Test Tube, Iodine solution,
etc.,
Procedure:
1. Peel and cut a potato into small cubes.
2. Measure the exact weight of the given potato.
3. Form the potato into a paste with the use of mortar and pestle and add the appropriate
amount of water.
4. Transform the paste into a beaker and enough water.
5. The homogenate should next be filtered through a cotton cloth to eliminate any particulates.
6. Allow the filtrate to settle for a while.
7. Gradually the starch lay at the bottombeaker.
8. Decant the starch-free supernatantcarefully.
9. Wash 3-4 times and decant the supernatant.
10. Gather the compacted starch mass and set it aside to dry.
11. Record the final weight of isolated starch and calculate the yield.

Iodine Test:
● Add a drop of 1 N HCl to a tiny amount of test solution.
● Add two drops of iodine solution to the mixture.
● The presence of starch is indicated by the production of a blue hue.
Result:
The supplied sample has 70 grams of starch per 100 grams of potato.
Discussion
In this experiment, it was discovered that starch could be isolated from potatoes. An iodine test was
done to authenticate the presence of starch in the sample by adding iodine solution to the starch
sample, which caused the sample to become blue. Because the starch contains amylose, the iodine
can slant into the amylose coil, causing the color change. The KI Reagent Iodine reagent is made
by dissolving iodine in water in the presence of potassium iodide since it is not particularly soluble
in water. This produces a soluble linear triiodide ion complex, which glides into the starch coil and
produces a vivid blue-blackcolor.

Experiment 4: Isolation of Casein and Lactose from Milk
Introduction
Milk is the most nutritionally complete food found in nature. All kinds of milk, human or animal,
contain vitamins (principally thiamine – VitB1, riboflavin – VitB2, pantothenic acid – VitB5, and
vitamins A, B12, D), minerals (calcium, potassium, sodium, phosphorus, and trace metals),
proteins (mostly casein), carbohydrates (principally lactose), and lipids (fats).
Whole milk is an oil-in-water emulsion, containing its 3.9% fat dispersed as micronized
globules. The fat emulsion is stabilized by complex phospholipids and proteins that are adsorbed
on the surfaces of the globules. Because the fat in milk is so finely dispersed, it is digested more
easily than fat from any other source. The globules are lighter than water, thus coalesce on
standing and eventually rise to the surface of the milk as cream. Vitamins A and D are fat-soluble
substances and are thus concentrated in the cream. The fats in milk are primarily triglycerides,
which are esters of saturated and unsaturated carboxylic acids with glycerol, a tri-alcohol [1].
Casein, the main protein in milk, is a phosphoprotein. The phosphate groups are attached to the
hydroxyl groups of some of the amino acid side chains. Casein exists in milk as the calcium salt,
calcium caseinate. It is actually a mixture of at least three similar proteins which differ primarily
in molecular weight and the amount of phosphorus groups they contain (α, β and κ caseins), they
form a micelle, or a solubilized unit. Neither the α nor the β casein is soluble in milk and neither
is soluble either singly or in combination. If κ casein is added to either one, or to a combination
of the two, however, the result is a casein complex that is soluble owing to the formation of the
micelle. A structure proposed for casein micelle is shown below[2]:
Figure 1. Casein

Calcium caseinate has an isoelectric point of pH 4.6. Therefore, it is insoluble in solutions of pH
less than 4.6. The pH of milk is about 6.6; therefore, casein has negative charge at this pH and is
solubilized as a salt. If acid is added to milk, the negative charges on the outer surface of the
casein micelles are neutralized (by protonation of the phosphate groups) and the neutral protein
precipitates, with the calcium ions remaining in solution:
Ca-caseinate + 2H+
→ casein + Ca2+
A natural example of this process occurs when milk sours. The souring of milk is an intricate
process started by the action of microorganisms on the principal carbohydrate in milk, lactose.
The microorganisms hydrolyze the lactose into glucose and galactose. Once galactose has been
formed, lactobacilli, a strain of bacteria present in milk, converts it to the sour-tasting lactic acid.
Since the production of the lactic acid also lowers the pH of the milk, the milk clots when it
sours due to the precipitation of casein. The structure of lactic acid is shown below [1]:
Figure 2. Lactic acid
When the fats and proteins have been removed from milk, the carbohydrates remain in the whey,
as they are soluble in aqueous solution. The main carbohydrate in milk is lactose.
Figure 3. Lactose
Lactose (4-O-(β-D-glucopyranosyl)-D-glucopyranose) is the only carbohydrate that mammals
synthesize. It is a disaccharide consisting of one molecule of D-glucose and one molecule of

D-galactose joined in 1, 4’fashion, and is synthesized in the mammary glands. In this process,
one molecule of glucose is converted to galactose and joined to another of glucose [2, 3].
Chemicals
Milk, Acetic Acid CH3COOH, Ethyl ether C4H10O, Ethanol C2H5OH
PROCEDURE
i. Isolation of Casein
Weigh out 5 grams of powdered nonfat dry milk and dissolve it in 20 mL of warm water in a 100
mL beaker. Bring the temperature of the solution to 55o
C (do not exceed) on a hot plate, remove
the thermometer, and then add dropwise a solution of 10% acetic acid while stirring with a
stirring rod. Do not add the acid too rapidly. Continue the acid addition (slightly less than 2
mL will be required), keeping the beaker on the hot plate, until the liquid changes from milky to
almost clear and the casein no longer separates. It is important not to add too much acid, because
it may hydrolyze some of the lactose in the milk and reduce your yield in isolation of lactose.
Stir the precipitated casein until it forms a large amorphous mass; then remove it with a stirring
rod or tongs and place it in another beaker.
Immediately add 0.75 grams calcium carbonate to the original beaker containing the remaining
liquid, stir for a few minutes, and save the resulting mixture for the later separation of lactose
below. The separation of lactose should be done as soon as possible during the same laboratory
period.
Collect the casein by suction filtration to remove as much water as possible. Press the solid with
a spatula. Place the casein in a 100 mL beaker and add 5 mL of a mixture of 1:1 ethyl ether and
ethanol (CAUTION: HIGHLY FLAMMABLE - NO FLAMES). Stir the casein in the ether
for a few minutes, decant the ether, and repeat the process with a second 5 mL portion of ether.

After the second washing with ether, suction filters the product. The ether washings remove any
small quantities of fat that may have precipitated with the casein. Place the casein between
several layers of paper towels to help dry the product, and let it stand in the air for 10-15
minutes. Divide the wet product in half, and weigh the two portions. Place one portion in a 125
mL Erlenmeyer flask with 35 mL of water and 0.5 mL of 1M NaOH, stopper the mixture, shake
it to ensure solution of as much of the casein as possible, and save it for use in the chemical tests
below. (You may carry out the chemical tests for the protein during this lab period if you have
time, or in your next lab period.) Allow the second portion to dry in your locker over the
following two weeks. When dry, weigh this portion and calculate the total yield of casein from
the powdered milk. Show your calculations.
ii. Isolation of Lactose
Gently boil the original liquid to which the calcium carbonate was added after isolation of casein.
Bumping will not be a problem so long as you stir the solution constantly and vigorously with a
glass rod. The solution will foam somewhat as it refluxes. This procedure precipitates the
remaining proteins lactalbumin and lactoglobulin. Suction filters the hot mixture to remove the
proteins and calcium carbonate, and transfer the hot, slightly yellow filtrate to a 125 mL
Erlenmeyer flask. Concentrate the filtrate to a volume of about 5 mL by heating with constant
swirling, again being careful to avoid bumping. Foaming can be controlled by heating the liquid
less vigorously and gently blowing onto it.
To the hot, concentrated solution, add 25 mL of hot 95% ethanol and 0.2 gram of decolourizing
carbon. Put this mixture aside and prepare a slurry of about 1 gram of Celite and 7.5 mL of 95%
ethanol. Suction filter the slurry into a Hirsch funnel containing a correct sized filter paper to
obtain a filter pad of Celite, and discard the alcohol in the filter flask. [The Celite filter pad helps
collect the very fine particles of carbon and prevents the normal filter paper from becoming
clogged.]
To the slightly cooled ethanol mixture containing the lactose, add 1 mL water. Suction filters the
mixture through the Celite filter pad, making sure the filtrate is clear. If the filtrate is cloudy, heat

it up and add another 0.5 mL of water. Transfer the filtrate to a 125 mL Erlenmeyer flask, heat it
until it clears, then allow to cool slowly. Stopper the flask and allow it to stand in your locker
until your next lab period.
Collect the crystals of lactose by suction filtration, and wash the product with a small amount of
cold 95% ethanol. Thoroughly dry the lactose and determine its weight and melting point.
Determine the percentage yield of lactose from the powdered milk, and show your calculations.
Discussion
Casein and lactose were extracted from milk in this experiment. Casein is precipitated by lowering
the pH of the milk to an acidic enough level to leave the protein insoluble while avoiding over-
acidification, which would cause the lactose to hydrolyze. In this situation, acetic acid was utilized.
Although the other proteins in acidic solution remain water-soluble, they may be precipitated and
separated by simply boiling and filtering the acidic solution. Isolated casein is soluble in alkaline
and acidic solutions but not in water, alcohol, or ether. After removing the casein, the excess acetic
acid is neutralized with calcium carbonate and the solution is heated to boiling to precipitate the
initially soluble protein, albumin. An ethanol derivative is ethyl ether. Because ethanol has a lower
polarity than It lowers the medium's dielectric constant, enhancing electrostatic interactions
that are crucial for casein micelle stability. The increased solubility of colloidal calcium
phosphate during acidification also assisted micellar dissociation.
The identification was accomplished through the use of a few critical chemical tests.
The biuret test is one of the most general protein assays. Protein in the sample reacts with
copper (II) sulfate. A positive test is the production of a violet-colored copper complex. The
presence of proteins was revealed by the formation of a violet hue.
* The xanthoprotein test: This is a typical response of proteins containing phenyl rings in
which concentrated nitric acid combines with the phenyl ring to produce a yellow-colored
product.
nitro aromatic compound Alkali addition at this The color will develop to orange at this
stage. The xanthoprotein reaction causes the yellow stains on the skin induced by nitric
acid.
Melting Point Determination
1. Melting point of casein: 280 °C
2. Melting point of Lactose: 340 °C
3. Percentage yield:
Percentage of product = amount of product recovered x 100%
Weight of milk

Weight of Casein = 0.570g
Weight of Lactose= 0.982g
Percentage of Casein= 0.590/ 5x 100= 11.8%
Percentage of Lactose= 0.955/ 5x 100= 19.1%
Why do we try to keep the temperature around 55°C?
Because particular hydrolytic transformation occurs when casein is subjected to the influence of
high temperature including

Experiment 5: Isolation of Citric acid from Lemon
Aim:
To isolate the citric acid from lemon juice using acid-base and precipitation reactions.
Apparatus and chemicals required:
1. Beaker, glass rod, funnel, filter paper, measuring cylinder, Buchner funnel, Buchner flask,
vacuum pump and water bath.
2. Lemon juice, sodium hydroxide, calcium chloride dihydrate, pH paper and sulphuric
acid.
Introduction:
Citric acid exists in greater amounts in citrus fruits. Lemons and limes have particularly high
concentrations of the acid. It can constitute as much as 8% of the dry weight of these fruits. In
olden days, industrial-scale citric acid was produced by treating the citrus fruit juice with
calcium hydroxide to precipitate calcium citrate. Calcium citrate was isolated and converted back
to the citric acid using dilute sulfuric acid. Citric acid can exist either in an anhydrous form or as
a monohydrate. The anhydrous form crystallizes from hot water, while the monohydrate forms
when citric acid is crystallized from cold water. The monohydrate can be converted to the
anhydrous form at about 78 °C. It decomposes with loss of carbon dioxide above about 175 °C.

Citric acid is normally considered to be a tribasic acid, with pKa values, extrapolated to zero
ionic strength, of 2.92, 4.28, and 5.21 at 25 °C. The pKa of the hydroxyl group has been found,
by means of 13C NMR spectroscopy, to be 14.4. The solution of citric acid and its salts can act
as buffer solutions between about pH 2-8. In biological systems around pH 7, the two species
present are the citrate ion and mono-hydrogen citrate ion. The citrate ion forms complexes with
metallic cations. The stability constants for the formation of these complexes are quite large
because of the chelate effect. Consequently, it forms complexes even with alkali metal cations.
However, when a chelate complex is formed using all three carboxylate groups, the chelate rings
have 7 and 8 members, which are generally less stable thermodynamically than smaller chelate
rings. In consequence, the hydroxyl group can be deprotonated, forming part of a more stable 5-
membered ring, as in ammonium ferric citrate. Citrate is an intermediate in the TCA cycle
(TriCarboxylic Acid cycle, or Krebs cycle), a central metabolic pathway for animals, plants, and
bacteria. Citrate synthase catalyzes the condensation of oxaloacetate with acetyl CoA to form
citrate. Citrate then acts as the substrate for aconitase and is converted into aconitic acid. The
cycle ends with regeneration of oxaloacetate. This series of chemical reactions is the source of
two-thirds of the food-derived energy in higher organisms.
PROCEDURE
1. Lemon was cut into pieces and squeezed to get the juice.
2. Measured the amount of lemon juice using a measuring cylinder
3. Transferred the 150 mL of lemon juice into a clean 250 mL beaker and tested its pH (2 or 3)
4. Added 10% sodium hydroxide solution till the pH becomes >9. The solution became cloudy
and the colour changed to a deep orange colour.
5. Filtered the solution to a new clean 500 beaker under vacuum.
6. Added 30 % calcium chloride solution to the beaker solution. Mixed the solution thoroughly.
7. Boiled the solution for 45 minutes in a water bath to precipitate the insoluble calcium citrate.
8. Filtered the precipitate of white calcium citrate under vacuum. Dried it.
9. Weigh the amount of isolated calcium citrate.
10. Calculated the required amount of sulfuric acid to neutralize the calcium citrate. Diluted the
sulfuric acid with distilled water.
11. Suspended the calcium citrate in a minimum amount of water.
12. Slowly, add the dilute sulfuric acid to the suspended calcium citrate.
13. Filtered the solution into a clean 250 mL beaker and concentrated to a small volume in a hot
plate.
14. Keep the solution with cover for about 1 to 2 weeks to crystallise the dissolved citric acid.
15. Isolated the crystals of citric acid by filtration.
16. Weighed the isolated citric acid.
Results:
The amount of citric acid isolated from 150 mL of lemon juice was 7.3 g.

Discussion:
Lemon and lime juice, both fresh and concentrated, contain more citric acid per liter than ready-to-
drink grapefruit juice, ready-to-drink orange juice, and orange juice squeezed from the fruit. On an
ounce-for-ounce basis, ready-to-drink lemonade and those that require mixing with water have 6
times the citric acid of lemon and lime juice.
Experiment 5: Isolation of Vanillin from
Vanilla
Aim:
To isolate the vanillin from vanilla.
Principle:
Vanillin is the molecule responsible for the characteristic flavor and odor of vanilla. Vanillin is
naturally produced by vanilla plant however it is present in a quite small amount thus most of the
Vanillin which is used worldwide has synthetically produced the extraction of vanillin from
artificial vanilla extract. It is preferable to use artificial vanilla extract rather than natural one as
the natural one will contain much more stuff in addition to vanillin materials.
Materials Required:

200 milliliters of artificial vanilla extract, 150 milliliters of dichloromethane or diethyl
ether, saturated solution of sodium chloride, magnesium sulphate and separatory funnel.
Procedure:
At first, the vanilla extract is poured in the separatory funnel then 50 milliliters of
dichloromethane are added to the separatory funnel. The separatory funnel is then mixed
thoroughly but with care to avoid the formation of an emulsion in between the phases if this
happens, we will not have a proper separation of the organic and the aqueous phases. Once the
two layers are settled down the stopcock is opened and the aqueous phase is drained out and the
organic phases poured in a clean beaker. This process is repeated three more times using 50
milliliters of dichloromethane in each. The organic layer is quite dirty and it has a lot of water in
it. In order to clear things up we have to wash the solution with brine then 30 milliliters of brine
are added to the separatory funnel to wash the organic phase. The organic phase is then collected
in a clean beaker and then dried using anhydrous magnesium sulphate. The magnesium sulphate
will get hydrated removing the moisture from the organic phase. The organic phases are then
filtered and placed in an appropriate flask 10 milliliters of dichloromethane to wash the surface
of the magnesium sulphate in order to obtain maximum recovery of the vanillin. In the
meantime, a minimum amount of the solution is analyzed by TLC and is compared with a
standard sample of vanillin the TLC plate is then viewed under the 254 nanometers light. The
two spots on the TLC plate with the same RF value confirms that the solution contains vanillin.
The majority of the solvent is removed using simple distillation, some activated charcoal is
added to the receiving flask in order to eliminate any compound from the recycled solvent after
filtration the solvent could be recovered and reused.
Once it is completely dried the crude vanillin can be recrystallized by adding 20 milliliters of
distilled water, the beaker is gently heated until everything is completely dissolved. However, we
have to keep the temperature below 80℃ degrees if not the vanillin will melt which will make
the crystallization after cooling. The crystals were dried under vacuum.

Warning: Dichloromethane is highly volatile and extremely flammable any open flames should
be avoided the experiments should be performed in a fume hood or in a well-ventilated area
glove goggles and lab coat are absolutely mandatory.
Discussion:
Vanilla is a kind of plant. The bean (fruit) is usually used for flavoring, but it is also utilized
in the production of medication. Because vanilla extract may be costly, lab-produced vanillin
is commonly used as a vanilla replacement. Vanilla is utilized as a flavour in medicine
syrups in the production process. Vanilla's effect in sweet baked goods is similar to that of
salt in savory baked goods: it enhances all of the other tastes in the recipe. Cookies and cakes
without it tend to taste flat and boring. Vanilla extract is created by steeping entire vanilla
beans in an alcohol-water combination.
Experiment 6:Extraction of pectin from orange peel
Aim:
To extract pectin from fresh orange peel.
Materials, Solvents & Reagents: Vacuum drier, Distillation unit, Thermometer, Nylon cloth,
Demineralized water, Isopropanol, Acetone, Citric acid, pH meter, Fresh orange peels.
Introduction:
In the fruits of plants, pectin helps keep the walls of adjacent cells joined together. Immature
fruits contain the precursor substance protopectin, which is converted to pectin and becomes more
water-soluble as ripening proceeds. At this stage the pectin helps ripening fruits to remain firm and
retain their shape. Pectins are soluble in pure water. Monovalent cation salts of pectinic and
pectic acids are usually soluble in water; di- and trivalent cations salts are weakly soluble or
insoluble. Dry powdered pectin, when added to water, has a tendency to hydrate very rapidly,
forming clumps.
Pectin is a complex polysaccharide consisting mainly of methoxy esterified α, d-1, 4-
galacturonic acid units. Pectins are categorized according to their methoxy content and whether
they form gels quickly or slowly. Pectin is a hydrocolloid which is used as a food emulsifier,
gelling agent, thickener, and stabilizer. It is the preferred choice of most of the food processors
as fat or sugar replacer in low-calorie foods. Pectin is an important polysaccharide with

applications in foods, pharmaceuticals, and a number of other industries. Its importance in the food
sector lies in its ability to form gel in the presence of Ca2+ ions or a solute at low pH. In the
pharmaceutical industry, it is used to reduce blood cholesterol levels and gastrointestinal disorders.
Other applications of pectin include use in edible films, paper substitute, foams and plasticizers,
etc.
Description:
Pectin is found in nature in the middle lamella of plant cells & chemically it is a mixture of
polymers of uronic acids & their methyl esters, Pectin of pharmaceutical significance is obtained
from orange peels, sun flower heads and raw papaya.
Procedure:
1. Cut the peels into smaller pieces. Weigh about 200gms of the peels, wash thoroughly with
water & immerse in one liter of water.
2. Adjust the PH to 4.5 with the addition of citric acid. Heat with stirring at 80-900
c for about one
hour. Add filter aid and filter immediately while the solution is hot.
3. Cool the filtrate & pour it slowly into 3 volumes of acidic Isopropanol or acetone. Stir the
solution thoroughly for the preparation of pectin.
4. Filter through nylon cloth & wash it several times with small volumes of 70% Isopropanol or
Acetone in order to make it free from acidic ions.
5. Dry the product in vacuum dryer. Weigh & store in well closed container
.
6. The yield of pectin from fresh orange peel is 4-5% W/W on dried basis. It is yellowish white
powder with mucilaginous taste. Dissolve 1gm in 9ml water by heating. On cooling, it forms
a stiff jelly.
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