PART
(1 )
EXTRACTION AND
ISOLATION TECHNIQUES
An Overview of Extraction Techniques for Medicinal and
Aromatic Plants
A wide range of technologies is available for the extraction of active components and essential
oils from medicinal and aromatic plants. The choice depends on the economic feasibility and
suitability of the process to the particular situation.
Extraction, as the term is used pharmaceutically, involves the separation of medicinally active
portions of plant or animal tissues from the inactive or inert components by using selective
solvents in standard extraction procedures.
General Methods of Extraction of Medicinal Plants:
1. Maceration
In this process, the whole or coarsely powdered crude drug is placed in a stoppered
container with the solvent and allowed to stand at room temperature for a period of at least 3
days with frequent agitation until the soluble matter has dissolved. The mixture then is strained,
the marc (the damp solid material) is pressed, and the combined liquids are clarified by filtration
or decantation after standing.
2. Infusion
Fresh infusions are prepared by macerating the crude drug for a short period of time with
cold or boiling water. These are dilute solutions of the readily soluble constituents of crude
drugs.
3. Digestion
This is a form of maceration in which gentle heat is used during the process of extraction.
It is used when moderately elevated temperature is not objectionable.
4. Decoction
In this process, the crude drug is boiled in a specified volume of water for a defined time;
it is then cooled and strained or filtered. This procedure is suitable for extracting water-soluble,
heat-stable constituents.
5. Percolation
This is the procedure used most frequently to extract active ingredients in the preparation
of tinctures and fluid extracts. A percolator (a narrow, cone-shaped vessel open at both ends) is
generally used.
6. Hot Continuous Extraction (Soxhlet)
In this method, the finely ground crude drug is placed in a porous bag or “thimble” made
of strong filter paper, which is placed in chamber E of the Soxhlet apparatus. The extracting
solvent in flask A is heated, and its vapors condense in condenser D. The condensed extractant
drips into the thimble containing the crude drug, and extracts it by contact. When the level of
liquid in chamber E rises to the top of siphon tube C, the liquid contents of chamber E siphon
into flask A. This process is continuous and is carried out until a drop of solvent from the siphon
tube does not leave residue when evaporated. The advantage of this method, compared to
previously described methods, is that large amounts of drug can be extracted with a much
smaller quantity of solvent.
7. Ultrasound Extraction (Sonication)
The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to
2000 kHz; this increases the permeability of cell walls and produces cavitation.
8. Supercritical Fluid Extraction
Supercritical fluid extraction (SFE) is an alternative sample preparation method with general
goals of reduced use of organic solvents and increased sample throughput. The factors to
consider include temperature, pressure, sample volume, analyte collection, modifier (coslvent)
addition, flow and pressure control, and restrictors. Generally, cylindrical extraction vessels are
used for SFE and their performance is good beyond any doubt.
The collection of the extracted analyte following SFE is another important step:
significant analyte loss can occur during this step, leading the analyst to believe that the actual
efficiency was poor.
There are many advantages to the use of CO2 as the extracting fluid. In addition to its
favorable physical properties, carbon dioxide is inexpensive, safe and abundant. But while
carbon dioxide is the preferred fluid for SFE, it possesses several polarity limitations. Solvent
polarity is important when extracting polar solutes and when strong analyte-matrix interactions
are present. Organic solvents are frequently added to the carbon dioxide extracting fluid to
alleviate the polarity limitations. Of late, instead of carbon dioxide, argon is being used because
it is inexpensive and more inert.
The component recovery rates generally increase with increasing pressure or temperature:
the highest recovery rates in case of argon are obtained at 500 atm and 150° C.
The extraction procedure possesses distinct advantages:
i) The extraction of constituents at low temperature, which strictly avoids damage
from heat and some organic solvents.
ii) No solvent residues.
iii) Environmentally friendly extraction procedure.
Steps Involved in the Extraction of Medicinal Plants
In order to extract medicinal ingredients from plant material, the following sequential
steps are involved:
1) Size Reduction
The dried plant material is disintegrated by feeding it into a hammer mill or a disc
pulverized which has built-in sieves. The objective for powdering the plant material is to rupture
its organ, tissue and cell structures so that its medicinal ingredients are exposed to the extraction
solvent. Furthermore, size reduction maximizes the surface area, which in turn enhances the
mass transfer of active principle from plant material to the solvent.
2) Extraction
Extraction of the plant material is carried out in three ways:
-
Cold aqueous percolation
-
Hot aqueous extraction (decoction)
-
Solvent extraction (cold or hot)
3) Filtration
The extract so obtained is separated out from the marc (exhausted plant material) by allowing
it to trickle into a holding tank through the built-in false bottom of the extractor, which is
covered with a filter cloth. The marc is retained at the false bottom, and the extract is received in
the holding tank. From the holding tank, the extract is pumped into a sparkler filter to remove
fine or colloidal particles from the extract.
4) Concentration
The enriched extract from percolators or extractors, known as miscella, is fed into a wiped
film evaporator where it is concentrated under vacuum to produce a thick concentrated extract.
The concentrated extract is further fed into a vacuum chamber dryer to produce a solid mass free
from solvent. Or the concentration could be done using Rotary Evaporation which is a technique
that employs a rotary evaporator (also called a “rotavap”) in order to remove excess solvents
from samples by applying heat to a rotating vessel at a reduced pressure. An important concept
that this technique applies is that liquids boil when the vapor pressure is equal to the external
pressure or atmospheric pressure. The machine utilizes a lower pressure than atmospheric
pressure which allows solvents to boil at lower temperatures. Furthermore, the rotation increases
the surface area and therefore evaporation proceeds more rapidly.
Essential Oils extraction techniques:
Essential oils are used in a wide variety of consumer goods such as detergents, soaps,
toilet products, cosmetics, pharmaceuticals, perfumes … etc. The world production and
consumption of essential oils and perfumes are increasing very fast. Production technology is an
essential element to improve the overall yield and quality of essential oil. The traditional
technologies pertaining to essential oil processing are of great significance and are still being
used in many parts of the globe.
Major constituents of essential oils are hydrocarbons, esters, terpenes, lactones, phenols,
aldehydes, acids, alcohols, ketones, and esters.
Methods of Producing Essential Oils
1. Hydrodistillation
In order to isolate essential oils by hydrodistillation, the aromatic plant material is packed in a
still and a sufficient quantity of water is added and brought to a boil.
Hydrodistillation of plant material involves the following main physicochemical processes:
i) Hydrodiffusion
ii) Hydrolysis
iii) Decomposition by heat
There are three types of hydrodistillation for isolating essential oils from plant materials:
a. Water distillation
b. Water and steam distillation
c. Direct steam distillation
2. Essential Oil Extraction by Hydrolytic Maceration Distillation
Certain plant materials require maceration in warm water before they release their essential oils,
as their volatile components are glycosidically bound. For example, leaves of wintergreen
(Gaultheria procumbens) contain the precursor gaultherin and other similar examples include
brown mustard (sinigrin), bitter almonds (amygdalin) and garlic (alliin).
3. Essential Oil Extraction by Expression
Expression or cold pressing, as it is also known, is only used in the production of citrus oils. The
term expression refers to any physical process in which the essential oil glands in the peel are
crushed or broken to release the oil.
4. Essential Oil Extraction with Cold Fat (Enfleurage)
The principles of enfleurage are simple. Certain flowers (e.g. tuberose and jasmine) continue the
physiological activities of developing and giving off perfume even after picking. Every jasmine
and tuberose flower resembles a tiny factory continually emitting minute quantities of perfume.
Fat possesses a high power of absorption and, when brought in contact with fragrant flowers,
readily absorbs the perfume emitted. This principle, methodically applied on a large scale,
constitutes enfleurage. During the entire period of harvest, which lasts for eight to ten weeks,
batches of freshly picked flowers are strewn over the surface of a specially prepared fat base
(corps), let there (for 24 h in the case of jasmine and longer in the case of tuberose), and then
replaced by fresh flowers. At the end of the harvest, the fat, which is not renewed during the
process, is saturated with flower oil. Thereafter, the oil is extracted from the fat with alcohol and
then isolated.
Isolation of Euginol from Clove Oil
Introduction:
Clove oil belongs to a large class of natural products called the essential oils. Many of
these compounds are used as flavorings and perfumes and, in the past, were considered to be the
“essence” of the plant from which they were derived.
Cloves are the dried flower buds of the clove tree, Eugenia caryophyllata, found in India
and other locations in the Far East. Steam distillation of freshly ground cloves results in clove
oil, which consists of several compounds. Eugenol is the major compound, comprising 85‐90 %.
Eugenol acetate comprises 9‐10 %. These structures are shown in Figure 1.
Eugenol contains a carbon‐carbon double bond and an aromatic hydroxyl group called a
phenol. These functional groups provide the basis for simple chemical tests used to characterize
the clove oil.
The boiling point of eugenol, an oil found in cloves, is 248 °C, but it can be isolated at a
lower temperature by performing a co-distillation with water, this process is also know as a
steam distillation.
Since eugenol is not soluble in water, the concentration of the eugenol in the vapor over
the boiling eugenol–water suspension does not depend on concentration of the eugenol. The
relative amounts of eugenol and water in the vapor simply depend on the vapor pressures of the
pure materials. The vapor pressure of water at 100 °C is 760 torr, and the vapor pressure of
eugenol at 100 °C is approximately 4 torr; therefore, the vapor is roughly 0.5 % eugenol. (Note,
the suspension boils when it’s vapor pressure is equal to the external pressure. Since both the
eugenol and the water are contributing to the vapor pressure of the suspension, the suspension
will boil before either pure substance would normally boil.)
Since the distillate will contain both water and eugenol, the eugenol must be extracted
from the water using an organic solvent. Once the eugenol is extracted into an organic solvent,
the organic layer is separated from the aqueous layer and dried. The eugenol is finally isolated by
evaporation of the organic solvent.
In practice, steam distillation is usually carried out by one of two methods. In the first
method, an excess amount of water is added to the compound of interest in a distilling flask.
The mixture is then heated to the boiling point. The resulting vapor is condensed and collected in
a receiving flask. The compound of interest is then separated from water, often by extraction. In
the second method, steam is bubbled into the compound of interest to effect the distillation. In
this experiment, the first method will be used because it is easier to set up.
Eugenol contains a carbon‐carbon double bond and an aromatic hydroxyl group called a
phenol. These functional groups provide the basis for simple chemical tests used to characterize
the clove oil. A solution of bromine (Br2) in chloroform decolorizes as Br2 reacts with the
double bond to form a colorless compound, as shown in Equation 3. A positive test is the
disappearance of the Br2 color.
A potassium permanganate (KMnO4) solution can oxidize a double bond at room
temperature to form a 1,2‐diol with the simultaneous reduction of Mn7+ in manganese oxide
(MnO2), as shown in Equation 4. A positive test is the disappearance of the purple KMnO4 and
the appearance of MnO2 as a brown precipitate.
Phenols (ArOH) react with the Fe3+ ion in iron(III) chloride (FeCl3) to give complexes
that arevblue, green, red, or purple, as shown in Equation 5. The color may last for only a few
seconds or for many hours, depending on the stability of the complex.
In this experiment, you will steam distill clove oil from freshly ground cloves. Following
the distillation, clove oil and water will be present in the receiving flask. Because clove oil will
be a minor fraction of the distillate, the clove oil must be extracted from the water into an
organic solvent such as dichloromethane. Removing dichloromethane leaves clove oil as the
product.
Experimental Procedure:
Part A: Isolation of Clove Oil
1) Weigh 50 g of dry cloves. Grind them to a coarse powder using a mortar and pestle.
Reweigh the powder and record the weight.
2) Transfer the ground cloves to a 1000 mL round‐bottom flask. Add 500 mL of distilled
water and a few boiling chips.
3) Assemble the water distillation apparatus – Clevenger apparatus – oil heavier than water
(Figure bellow).
4) Stop the distillation after 3-4 hours.
5) Allow the distillate to cool to room temperature. Carefully pour the distillate into a
separatory funnel. Add 10 mL of saturated NaCl solution.
6) Rinse the inside of the condenser and the receiver with 5‐10 mL of CH2Cl2 into the
separatory funnel.
7) Cap the separatory funnel and gently swirl the contents for several seconds. Vent the
separatory funnel frequently. After the pressure has been vented, shake the contents
vigorously to thoroughly mix the two layers.
8) Allow the layers to separate. Drain the CH2Cl2 layer into an Erlenmeyer flask.
9) Repeat the extraction of the aqueous layer twice, each time with 5 mL portion of
CH2Cl2. Combine organic layer in the same Erlenmeyer flask.
10) Dry the combined CH2Cl2 solution with anhydrous Na2SO4.
11) Decant the CH2Cl2 solution into a pre‐weighed ceramic evaporating dish, making certain
that no Na2SO4 is transferred with the solution.
12) Place the evaporating dish on a hot water bath to remove CH2Cl2. When all of the
CH2Cl2 has been evaporated, allow the evaporating dish to cool to room temperature.
Weigh it to the nearest 0.001 g and record the weight. Subtract the mass of the empty
dish to obtain the mass of the clove oil. Report the weight and percent yield of clove oil
to your instructor.
Part B: Characterization of the Isolated Clove Oil
1- Dissolve the clove oil in 2‐3 mL of methanol.
2- Obtain six test tubes and label them 1‐6. Label tubes 2, 4, and 6 as “blank”. Add 1 mL of
methanol to all 6 test tubes.
3- Add 5 drops of clove‐oil solution to tubes 1, 3, and 5. Gently swirl each tube.
4- Add 5 drops of bromine in chloroform to tubes 1 and 2. Gently swirl and record your
observation.
5- Add 5 drops of KMnO4 solution to test tubes 3 and 4. Gently swirl and record your
observation.
6- Add a few drops of FeCl3 solution to test tubes 5 and 6. Gently swirl and record your
observation.
Part C: Isolation of euginol from clove oil
1) Dissolve the oil in dichloromethane to 50 ml the add 30 ml KOH solution 5 % in
separatory funnel
2) Save the aqueous layer in flask and repeat the addition of 25 ml of KOH solution twice.
3) Combine the aqueous solutions back to separatory funnel and wash with pure CH2Cl2
4) Transfer the aqeous layer to separatory funnel 250 ml then acidify to pH 1 using 5 % HCl
dropwise while stirring. Test pH using pH universal paper. Notice the formation of
cloudy layer. (why?????)
5) Add about 20 ml dichloromethane to the separatory funnel. Save the organic layer and
extract again with CH2Cl2 twice.
6) Using the same separatory funnel wash the combined dichloromethane portions with pure
15 ml distilled water.
7) Transfer the organic layer to clean dry beaker. Add 15 gm of anhydrous sodium sulfate to
the beaker. (why????)
8) Dry the organic solvent to get pure euginol.
9) After dryness weight the euginol and calculate the percentage yield which equals =
Wt. of euginol / wt. of clove powder * 100 %
And
The percent of euginol in oil.
10) Identify the isolated euginol by TLC using dichloromethane as mobile phase and euginol
and clove oil standards.
Isolation of Hesperidin from Orange Peel Using
Soxhlet Extractor
Introduction:
Hesperidin, a polyphenolic bioflavonoid, is the predominant flavonoid in orange peel and
other citrus fruits. The highest concentration of hesperidin can be found in the white parts and
pulps of the citrus peels. Hesperidin can also be found in green vegetables. Hesperidin is a
flavanone glycoside consisting of the flavone hesperitin bound to the disaccharide rutinose. The
sugar causes hesperidin to be more soluble than hesperitin. Figure below shows its structure.
Hesperidin is an antioxidant that enhances the action of vitamin C to lower cholesterol levels. It
is also known to have pharmacological action as an anti-inflammatory, antihistaminic, and
antiviral agent.
Hesperidin alone, or in combination with other citrus bioflavonoids (diosmin, for
example), is most often used for blood vessel conditions such as hemorrhoids, varicose veins,
and poor circulation (venous stasis). It is also used to treat lymphedema, a condition involving
fluid retention that can be a complication of breast cancer surgery. The molecular mechanism of
the inhibitory effect of hesperidin on bone resorption is not clear.
Hesperidin improves the health of capillaries by reducing the capillary permeability so
that it could treat leg ulcers caused by poor circulation, when used in combination with diosmin.
Hesperidin, in combination with diosmin and a compression dressing, seems to improve
healing of small ulcers (less than 10 cm) caused by poor blood circulation. Hesperidin is used to
reduce hay fever and other allergic conditions by inhibiting the release of histamine from mast
cells. The possible anti-cancer activity of hesperidin could be explained by the inhibition of
polyamine synthesis.
The following doses have been studied in scientific research:
-
For the treatment of hemorrhoids inside the anus: 150 mg of hesperidin plus 1350 mg of
diosmin twice daily for 4 days, followed by 100 mg of hesperidin and 900 mg of diosmin
twice daily for 3 days.
-
For preventing the return of hemorrhoids inside the anus: 50 mg of hesperidin plus 450
mg of diosmin twice daily for 3 months.
-
For the treatment of leg ulcers caused by poor blood circulation (venous stasis ulcers): a
combination of 100 mg of hesperidin and 900 mg of diosmin daily for up to 2 months.
Hesperidin can be isolated by two different methods:
-
The first method involves extracting the dried citrus peel successively with petroleum
ether followed by methanol. The petroleum ether removes the essential oils in the peel
and the methanol will extract the glycoside (hesperidin).
-
The second method uses an alkaline extraction of chopped orange peel and acidification
of the extract. The hesperidin can then be crystallized from the acidified extract. Because
of its highly insoluble, crystalline nature, hesperidin is one of the easiest flavonoids to
isolate.
Work Procedure:
1) 150 mL petroleum ether (40 – 60°C) are filled in a 250 mL round bottom flask with
magnetic stir bar.
2) 50g dried and powdered orange peel are placed in the extraction sleeve of a Soxhlet
extractor and covered with a little glass wool.
3) A reflux condenser is put on the Soxhlet extraction unit, and then the reaction mixture is
stirred and heated for 4 hours under strong reflux.
4) The petroleum ether extract is discarded. In order to remove the adherent petroleum
ether, the content of the extraction sleeve is laid out in an extensive crystallisation dish.
5) Afterwards the substance is placed again in an extraction sleeve and, like before, but with
150 mL methanol, extracted unless the solvent leaving the extraction sleeve is colourless
(1 to 2 hours).
6) The extract is evaporated at the rotary evaporator until syrup consistency is reached. The
residue is mixed with 50 mL of 6% acetic acid; the precipitated solid is the crude
hesperidine.
7) It is sucked off with a Buechner funnel, washed with 6% acetic acid and dried 60 °C until
it is constant in weight.
8) For recrystallization, a 5% solution of the crude product in dimethyl sulfoxide is
produced under stirring and heating to 60–80 °C. Afterwards the same amount of water is
added slowly whilst stirring. When cooling to room temperature the hesperidine
precipitates. It is sucked off, first washed with little warm water and then with isopropanol and dried in the desiccators until it is constant in weight.
Analytics
Reaction control with TLC
TLC-Conditions:
adsorbant: TLC plates GF254 normal silica
Mobile phase: n-butanol : acetic acid : water = 4 : 1 : 5
Rf (hesperidine) = 0.6
Extraction Scheme:
PART (2)
PHYTOCHEMICAL
SCREENING OF
UNKNOWN DRUGS
PRELIMINARY PHYTOCHEMICAL SCREENING
OF UNKNOWN DRUGS
Phytochemistry is mainly concerned with enormous varieties of secondary plant
metabolites which are biosynthesized by plants. Most of the best plant medicines are the sum of
their constituents.
The beneficial physiological and therapeutic effects of plant materials typically result
from the combinations of these secondary products present in the plant. The information on the
constituents of the plant clarifies the uses of the plants but only a small percentage have been
investigated for their phytochemicals and only a fraction has undergone biological or
pharmacological screening.
In phytochemical evaluation the powdered leaves were subjected to phytochemical
screening for the detection of various plant constituents, characterized for their possible bioactive
compounds, which have been separated and subjected to detailed structural analysis.
Phytochemical analysis procedure:
 After authentification, the fresh, healthy plant dry in shade for 2-3weeks then
pulverize in a blender, sieve and use for further studies.
 Subject the powdered drugs under investigation to the following tests:
1. Detection of alkaloids:
Dissolve the extract individually in dilute Hydrochloric acid and filter.
a) Mayer’s Test: Treat the filtrate with Mayer’s reagent (Potassium Mercuric Iodide).
Formation of a yellow colored precipitate indicates the presence of alkaloids.
b) Wagner’s Test: Treat the filtrate with Wagner’s reagent (Iodine in Potassium Iodide).
Formation of brown/reddish precipitate indicates the presence of alkaloids.
c) Dragendroff’s Test: Treat the filtrate with Dragendroff’s reagent (solution of
Potassium Bismuth Iodide). Formation of red precipitate indicates the presence of alkaloids.
2. Detection of carbohydrates:
Dissolve the extract individually in 5 ml distilled water and filter. Use the filtrate to test for
the presence of carbohydrates.
Molisch’s Test: Treat the filtrate with 2-3 drops of 1% alcoholic α-naphthol and 2ml of
concentrated sulphuric acid was added along the sides of the test tube. Formation of the violet
ring at the junction indicates the presence of Carbohydrates.
Benedict’s test: Treat the filtrate with Benedict’s reagent and heated gently. Orange red
precipitate indicates the presence of reducing sugars.
Fehling’s Test: Treat the filtrate with dil. HCl, neutralize with alkali and heated with
Fehling’s A & B solutions. Formation of red precipitate indicates the presence of reducing
sugars.
3. Detection of anthraquinone glycosides:
Extracts were hydrolysed with dil. HCl, and then subjected to test for glycosides.
Modified Borntrager’s Test: Treat the filtrate or powder with dil. HCl, ferric chloride
solution and immerse in boiling water for about 10 minutes. Cool and extract with equal volumes
of benzene or chloroform. The benzene layer will be separated and treated with ammonia
solution. Formation of rose-pink colour in the ammonical layer indicates the presence of
anthranol glycosides.
4. Detection of cardiac glycosides (Keller Kelliani’s test):
2ml of filtrate will be treated with 2ml of glacial acetic acid in a test tube and a drop of ferric
chloride solution will be added to it. This will be carefully underlayed with 1ml concentrated
sulphuric acid. A brown ring at the interface indicated the presence of deoxy sugar characteristic
of cardenolides. A violet ring may appear below the ring while in the acetic acid layer, a greenish
ring may form.
5. Detection of saponins glycosides:
Froth Test: 1 gm of powder will be diluted with distilled water to 6 ml and this will be
shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the
presence of saponins.
Foam Test: 0.5 gm of powder will be shaken with 2 ml of water. If foam produced persists
for ten minutes it indicates the presence of saponins.
6. Detection of unsaturated sterols and/or triterpenes:
Dissolve the residue in 10 ml of Petroleum ether or anhydrous chloroform and filter. Divide
the filtrate into two equal portions and test as follows:
Salkowski’s Test: Filtrate will be treated with with 2 ml of sulphuric acid, shaken and
allowed to stand. Appearance of golden yellow color changing to orange and then to red,
indicates the presence of unsaturated sterols and/or triterpenes.
Libermann Burchard’s test: To the chloroform solution, add 1 ml of acetic anhydride
followed by the addition of sulfuric acid down the walls of the test tube to form a layer, the
formation of a reddish-violet color at the junction of the two layers, and a green color in the
chloroform solution indicates the presence of unsaturated sterols and/or triterpenes.
7.
Detection of phenols:
Ferric Chloride Test: Extracts will be treated with 3-4 drops of ferric chloride solution.
Formation of bluish black colour indicates the presence of phenols.
8. Detection of flavonoids
Alkaline Reagent Test: 2ml of extract will be treated with few drops of 20% sodium
hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of
dilute hydrochloric acid, indicates the presence of flavonoids.
Lead acetate Test: Extracts were treated with few drops of lead acetate solution (10 %).
Formation of yellow color precipitate indicates the presence of flavonoids.
9. Detection of tannins:
Extract about 1 g of the powdered drug with 5 ml of alcohol and filter. Test the clear
alcoholic filtrate as follows:
a- Add ferric chloride, a blue color indicates the presence of pyrogallol (hydrolyzable)
tannins, and a green color indicates the presence of catechol (condensed or nonhydrolyzable) tannins.
b- To 5 ml of the alcoholic extract add 2 ml of vanillin-hydrochloric acid solution, a rose red
color indicates the presence of gallic acid.
c- To 5 ml of the alcoholic extract add 2 ml of freshly prepared bromine water, the
formation of a precipitate indicates the presence of tannins ofnon-hydrolysable type.
10. Detection of lignin:
2 ml of filtrate will be treated with alcoholic solution of phloroglucinol and concentrated
hydrochloric acid. Appearance of red colour shows the presence of lignin.
11. Detection of proteins and amino acids:
Ninhydrin Test: (1% ninhydrin solution in acetone): 2ml of filtrate will be treated
with 2-5 drops of ninhydrin solution placed in a boiling water bath for few minutes and observed
for the formation of blue to purple colour. Formation of the colour indicates the presence of
amino acid.