EXERCISE NO.

3
PHYTOCHEMICAL SCREENING OF CRUDE DRUGS

Intended Learning Outcomes:

At the end of the experiment, students should be able to:
1. Demonstrate skills in determining the active constituents available in plant crude extract
following the stated procedures indicated for various phytochemical screening;
2. Calculate the percentage yield of crude extract;
3. Compare and interpret the results of phytochemical tests done as performed by each group.

Pre-Task
1. The plant of choice by each group which was previously air dried serve as starting material for
this experiment provided the amount will not be less than 150 g.
2. Suitable wide-mouthed glass container with cover
3. Preparation of 80% ethanol (300-500 mL) per group

Materials and Methods:

Air-dried plant material Reflux condenser Calculator

Aluminium pan of suitable size / Casserole / Mettler/analytical balance
evaporating dish water bath with rings
Kitchen scissor/cutter Hot plate Dessicator
Roll of Aluminium foil Funnel Separatory funnel (with iron
stand, iron ring/clamp)
Muslin cloth/ filter paper Glass bottle/reagent Pipettes (1-, 2-, 5-mL) and
bottle rubber aspirator
Long forcep/crucible tong Bunsen burner Test Tubes /test tube holder
500 mL Erlenmeyer flask with fitted cork Dry Heat oven Beakers
Petri dish Cork borer Test tube rack
Glass (long nose) droppers spatula Glass rod
Litmus paper (blue/red) Cleaning agents

Formula for Essential Calculations:

(wt. of plant sample)

Equivalent weight per mL) =
(vol. of extract)

wt. of the extract

% yield of crude extract) = x 100
wt. of plant sample

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Reagents Needed for Phytochemical Procedures:

Please take note that some reagents will be freshly prepared prior to conduct of phytochemical
screening procedures.

Fehling’s reagent (Fehling’s A & B) Wagner’s reagent Sodium pricrate solution

Molisch’s reagent Valser’s reagent Sodium carbonate
Benedict’s reagent Dragendorff’s reagent Picric acid
Millon’s reagent Mayer’s reagent 1N sodium hydroxide
concentrated Nitric acid Anhydrous Sodium sulphate Sodium nitroferricyanide TS
5% NaOH (1 g of sodium nitroferricyanide in
water to make 20 mL)
Petroleum ether Acetic anhydride solution Magnesium oxide
Hexane concentrated Sulfuric acid 10% sodium chloride
28% ammonia Ferric chloride reagent 1% gelatin solution
Copper sulphate TS (mix 0.3 mL of 10% ferric chloride 1% sodium nitrite solution
solution with 50 mL glacial acetic
acid)
Chloroform Ammonia TS Gelatin-salt reagent
(1% gelatin solution + 10% sodium
chloride solution)
2M Hydrochloric acid Benzene 5% Ferric chloride solution
Sodium chloride 0.5N potassium hydroxide Magnesium ribbon
95% Ethanol diluted Hydrogen peroxide Octyl alcohol
80% Ethanol Glacial acetic acid

Reminder: Please take photos of plant material undergoing PHYTOCHEMICAL SCREENING

(Optional: one-minute file video of the group activity)

PROCEDURES Remarks/Observations
PREPARATION OF PLANT MATERIAL
1. Secure a clean, dry glass jar container.
2. Grind and pulverize the previously dried plant material using a blender
or osteorizer.
3. Weigh all the pulverized material. Set aside at least 100-150 g of the
material for maceration in a 500-mL Erlenmeyer flask. Keep and store
the rest of the pulverized material in appropriate container.
4. Pour 80% ethanol in an Erlenmeyer flask containing the plant
material, just enough to cover the sample (an inch above sample)
5. Macerate it for 2 to 3 day. Reflux distillation can be done.
6. Add enough 80% ethanol if the plant material swells and sucked up the
solvent. Stir mix the contents and expressed the material using muslin
cloth to collect the liquid extract.
7. Filter, combine the filtrates and discard the residue.

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8. Measure the volume of filtrate collected.
9. Reduce the filtrate under vacuum using rotary evaporator.
10. Concentrate further the extract in an evaporating dish under water
bath with a temperature not exceeding 60 deg C.
11. Measure the volume of the concentrated extract.
12. Store the extract in a suitable tightly closed glass jar/bottle container
and keep it in a cool temperature.
13. Label the bottle properly indicating the name of the extract,
equivalent weight per mL, date of extraction and class & group code.
14. Submit the summary of computed value of volume required for
equivalent weight of extract needed for all the succeeding tests.

PROCEDURES: Screening for CARBOHYDRATES Remarks/Observations

1. Evaporate the equivalent 10 g of plant extract to a dry or syrupy
consistency using water bath.
2. Dissolve the residue in 10 mL of distilled water and divide it into 4 test
tubes equally. Label the tubes A, B, C, and D (control).
FEHLING’S TEST
1. Boil 2.5 mL of Fehling’s reagent (equal parts of Fehling’s A & B) with
2.5 mL of extract contained in test tube A.
2. Observe for the presence of brick red precipitate.
*Note: Test tube in half-filled beaker of boiling water
MOLISCH’S TEST
1. Add 3 to 5 drops of α-naphthol solution to 2.5 mL of extract contained
in test tube B while shaking it carefully. Introduce slowly few drops of
conc. sulphuric acid in the test tube in a slant position. Note and
record the coloration formed.
*Note: Did you notice a violet ring or purple coloration formed at the
junction of two liquids?
BENEDICT’S TEST
1. To test tube C, add an equal amount of Benedict’s reagent and boiled
in water bath for 5 minutes. Observe and record color changes of
solution.
*Note: Did you see green, yellow or red coloration?
Teacher’s Note:

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PROCEDURES: Screening for PROTEINS Remarks/Observations
1. Evaporate the equivalent 10 g of plant extract to a dry or syrupy
consistency using water bath.
2. Dissolve the residue in 10 mL of distilled water and divide it into 3 test
tubes equally. Label the tubes A, B, C, and D(control).
MILLON’S TEST
1. Add 10 drops of Millon’s reagent to test tube labelled A and boil in
water bath for 1 minute. Cool
*Note: Did you see yellow precipitate?
2. Add 5 drops of 1% sodium nitrite. Heat slightly.
*Note: Is brick-red color formed?
XANTHOPROTEIC TEST
1. Add carefully 1 mL of nitric acid to test tube labelled B. Take note of
observed color or changes in the solution.
*Note: Did you see white precipitate?
2. Boil the solution and record any color changes.
*Note: Did you observe yellow coloration?
3. Cool the test tube and add 2 mL of 20% ammonia solution.
*Note: Did the solution turned to orange color?
BIURET TEST
1. To test tube labelled C, add 1 mL of 5% NaOH and 1 to 2 drops of
copper sulphate.
*Note: Did you see violet coloration?
Teacher’s Note:

PROCEDURES: Screening for ALKALOIDS Remarks/Observations

1. Evaporate the equivalent 2O g of plant extract to a dry or syrupy
consistency using steam bath.
2. Add 5 mL of 2M HCl, stir and heat it for about 5 minutes in a water
bath and cool.
3. Add 0.5 g NaCl, stir and filter.
4. Wash the residue with enough 2M HCl to bring the filtrate to a volume
of 8 mL.
PRELIMINARY TEST
5. Prepare 5 test tubes and label it as A, B, C, D, and E (control).
6. Test tubes labelled A, B, C, D will contain one mL of filtrate each while
test tube labelled E will contain 4 mL of the filtrate from STEP 4.
Reserve test tube labelled E for confirmatory test.

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7. Add 2 to 3 drops of alkaloidal reagents to the respective test tubes:
Test tube A – Mayer’s reagent
Test tube B – Wagner’s reagent
Test tube C – Valser’s reagent
Test tube D – Dragendorff’s reagent
*Note: Observe the relative amount of precipitation and record as
follows: (-) no turbidity;
(+) slight turbidity;
(++) definite turbidity; and
(+++) heavy precipitation
CONFIRMATORY TEST
1. Add enough 28% ammonia to the filtrate contained in test tube E until
the solution becomes alkaline to litmus.
*CAUTION: Ammonia causes burns and has extremely irritating vapors.
2. Extract the alkaline solution with few portions of chloroform (10 mL)
three times.
*Note: Separatory funnel can be used
*CAUTION: Chloroform is carcinogenic
3. Combine all chloroform extracts and reserve the aqueous layer for
TEST FOR QUATERNARY BASES and/or AMINES.
4. Evaporate the chloroform extract to dryness over a steam bath under
the hood.
5. Add 5 mL of 2M hydrochloric acid.
6. Filter the solution and divide it into 4 equal portions in test tubes
labelled A, B, C, and D.
7. Add 2 to 3 drops of alkaloidal reagents to the respective test tubes:
Test tube A – Mayer’s reagent
Test tube B – Wagner’s reagent
Test tube C – Valser’s reagent
Test tube D – Dragendorff’s reagent
*Note: Observe the relative amount of precipitation and record as
follows: (-) no turbidity;
(+) slight turbidity;
(++) definite turbidity; and
(+++) heavy precipitation
TEST FOR QUATERNARY BASES/AMINES
1. Acidify the alkaline aqueous layer obtain from STEP 3 under
CONFIRMATORY TEST with 2M hydrochloric acid.
2. Filter and divide it into 4 equal portions in test tubes labelled A, B, C,
and D.
3. Add 2 to 3 drops of alkaloidal reagents to the respective test tubes:
Test tube A – Mayer’s reagent
Test tube B – Wagner’s reagent

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 Test tube C – Valser’s reagent
Test tube D – Dragendorff’s reagent
*Note: Heavy precipitation (+++) will indicate the presence of quaternary
and/or amine oxide bases.
Teacher’s Note:

PROCEDURES: Screening for UNSATURATAED STEROLS/TRITERPENES Remarks/Observations

1. Evaporate the equivalent 2O g of plant extract to a dry or syrupy
consistency using water bath. Cool to room temperature.
2. Add about 10 mL of petroleum ether to the dish, stir for minutes and
allow to settle.
3. Decant and discard the supernatant liquid. Repeat the defatting
procedure until most of the pigments has been removed.
4. Add 10 mL of chloroform to the residue, stir thoroughly for 5 minutes.
5. Decant into a suitable tube passing over 100 mg anhydrous sodium
sulphate in a filter paper.
6. Divide the filtrate into three dry test tubes and label it A, B, and C.
*Note: Test tube C will serve as color control
LIEBERMANN-BURCHARD TEST
1. To test tube A, add 3 drops of acetic anhydride. Mix gently by swirling
the tube.
2. Add one drop of concentrated sulphuric acid and mix gently. Set aside
for an hour.
*Note: Observe for color changes immediately.
Compare it with Test tube C.
SALKOWSKI TEST
1. To test tube B in a slant position (450 angle), add 1 to 2 mL of
concentrated sulfuric acid running down slowly the inside walls of
test tube.
*Note: Did you see any immediate color changes at the junction of the
extract and the sulphuric acid?
2. Gently mix the sulphuric acid and extract. Observe for immediate /
gradual color changes over a period of one hour.
*Note: Is a cherry red color noticed?
KELLER-KILLIANI TEST (Screening for 2-deoxysugars)
1. Evaporate 10 mL of the 80% ethanolic extract to incipient dryness on a
steam bath.
2. Add 3 mL of the ferric chloride reagent, stir, mix well and transfer to a
test tube.

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3. Hold the test tube at 450 angle and run down slowly 1 mL of
concentrated sulphuric acid. Do not shake or agitate the test tube at
this point.
*Note: Did you notice the purple “ring” at the interface
Teacher’s Note:

PROCEDURES: Screening for FLAVONOIDS and LEUCOANTHOCYANINS Remarks/Observations

1. Take an equivalent of 10 g sample from the stock sample extract and
evaporate to incipient dryness over a steam bath.
2. Cool at room temperature.
3. Defat the residue with petroleum ether. Decant and discard the
supernatant liquid. Repeat the defatting procedure until most of the
pigments has been removed.
4. Treat the residue with 10 mL of 80% ethanol, mix and stir.
5. Filter and divide the filtrate into three test tubes labelled A, B, and C
respectively. Test tube C will serve as control.
Bate-Smith and Metcalf Test
1. Treat Test Tube A with 0.5 mL concentrated HCl (12M) and take note
of any observable color changes.
2. Heat the solution in water bath for 15 minutes. Remove from heat.
Observe for any further color developed within 45 minutes. Record the
result.
*Note: Did you see any strong red or violet coloration?
Wilstatter Cyanidin Test
1. To test tube B, add 0.5 mL concentrated HCl (12M).
2. Place 2 to 3 pieces of ½ inch magnesium ribbons. Observe for any color
change within 10 minutes.
3. If definite coloration occurs, dilute it with an equal volume of water
and add 1 mL of octyl alcohol. Shake the solution and stand it for 5
minutes or more. Note and record any color observed in each layer.
*Note: Did you see colors ranging from orange to red, crimson, and
magenta or to green or blue?
Teacher’s Note:

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PROCEDURES: Screening for CYANOGENIC GLYCOSIDES Remarks/Observations
1. Weigh at least 3 grams of your powderized plant sample. Transfer it in
a 125 mL Erlenmeyer flask and add enough distilled water to moisten
the sample.
2. Prepare a strip of sodium picrate paper:
Freshly prepared sodium picrate solution should be done first by
weighing 5 g of sodium carbonate and 0.5 g of picric acid and mixing it in
sufficient amount of distilled water to make 100 mL. (This will be
assigned to one or two members in the class).
Cut Whatman #1 filter paper into strips that will suitably fit in the flask.
Dip one strip into a solution of sodium picrate. Drip dry and finally blot
between sheets of absorbent paper towel.

3. Add 1 mL of chloroform to the flask containing the plant material.

4. Insert the piece of freshly prepared sodium picrate paper in the flask
just above the plant sample and fold over the rim of the flask. Make
sure it will not touch the inner sides of the flask or the plant material.
5. Stopper the flask and warm at 35 deg C or at room temperature for up
to three hours. Observe for any color change in the paper.
*Note: Did you see various shades of red colors?
Teacher’s Note:

PROCEDURES: Screening for SAPONINS Remarks/Observations

1. Take an equivalent weight of 20 g of plant extract and evaporate to
incipient dryness using steam bath.
2. Add 30 mL of hot saline solution to the residue stir well and continue
heating on water bath for 2 minutes.
3. Add 2 g of magnesium oxide; stir to mix for 5 minutes.
4. Filter (vacuum) the mixture and adding sufficient quantity of fresh
saline solution to make 20 mL. Divide the filtrate into 2 equal portions.
FROTH TEST
1. Transfer 10 mL of the filtrate to a 20-mL test tube and stopper it and
shake vigorously. Let it stand for 3 minutes and measure the height of
foam it produced. After 30 minutes standing, measure again the foam
height in cm.
*Note: Did you see persistent froth?

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HEMOLYSIS TEST
1. Prepare blood agar plate.
2. Bore 3 equidistant holes on the blood agar using a small test tube or
cork borer (10 mm diameter). Label the bottom of inverted plate with
marking pen.
3. Fill each hole with respective solution of your sample extract, gugo
extract and control saline solution.
4. Observe at the end of 3, 10, 15 and 30 minutes.
*Note: Did you see clear zone formation?
Teacher’s Note:

PROCEDURES: Screening for TANNINS AND POLYPHENOLS Remarks/Observations

1. Evaporate an equivalent 10 g of your plant extract to dryness on a
steam bath.
2. Take the residue with 20 mL of hot distilled water. Stir to cool.
3. Add 5 drops of 10% sodium chloride. Filter the solution.
4. Prepare 4 test tubes and introduce at least 3 mL of the filtrate in each
test tube labelled A, B, C, and D (control).
5. To test tube A, add 5 drops of 1% gelatin solution.
*Note: Did you see any white curdled formation?
6. To test tube B, add 5 drops of gelatin-salt reagent.
*Note: Did you form white precipitate?
7. To test tube C, add few drops of ferric chloride TS.
*Note: What color change reaction occurred?
8. Record all your observation.
Teacher’s Note:

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PROCEDURES: Screening for ANTHRAQUINONES Remarks/Observations
BORNTRAGER TEST
1. Evaporate an equivalent 10 g of your plant extract to dryness on a
steam bath.
2. Add 10 mL of distilled water to the defatted residue, mix well, and
filter into a small separatory funnel.
3. Add 10 mL of benzene, shake to mix well and allow the two phases to
separate.
4. Drain out the aqueous layer and transfer the benzene phase to a test
tube.
5. Introduce 5 mL of ammonia TS, mix well and observe the benzene
layer for color change
MODIFIED BORNTRAGER TEST
1. Evaporate an equivalent 10 g of your plant extract to dryness on a
steam bath.
2. To the residue, add 10 mL of 0.5N potassium hydroxide and 1 mL of 5%
hydrogen peroxide solution and heat it for 10 minutes over steam
bath. Cool and filter.
3. Acidify the filtrate with approximately 10 drops of glacial acetic acid.
4. Transfer the acidified solution into a separatory funnel and partition
with 10 mL of benzene.
5. Filter the benzene phase and transfer 5 mL to a test tube containing
2.5 mL of ammonia TS. Mix well and observe for color change.
6. Record all your observation.
Teacher’s Note:

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 Table 3.
Results of Phytochemical Screening

Remarks
Name of Test Actual Results Interpretation
(+ or -)

Post-Task

1. Each group will prepare a written ACTIVITY

REPORT on PHYTOCHEMICL SCREENING OF
CRUDE DRUGS using the format/template
issued to them by the instructor. Important
highlights include the following: Learning
Objectives, Methodology, Discussion of
Results and Interpretation, Conclusion, and
Recommendation.
2. Post-laboratory discussion will be held after
all the groups completed the activity as
scheduled.
3. Assessment quiz will be conducted on a
scheduled basis too.

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