This document summarizes various phytochemical screening tests that can be used to identify the presence of important plant constituents like carbohydrates, proteins, alkaloids, flavonoids, tannins, saponins, anthraquinones, and other metabolites. It provides details of common confirmatory tests used which involve visual observation of color changes or formation of precipitates when plant extracts are treated with specific detecting reagents. The results of these tests can help determine the major phytoconstituent classes present in a plant sample.

1. Phytochemical Screening
Abhishek Gupta
Associate Professor
Department of Pharmacognosy,
Hygia Institute of pharmaceutical Education & Research,
Lucknow, Uttar Pradesh, India
Test for Carbohydrates:
Molisch's reagent (a-NaphthoI): 15g of a-naphthol was dissolved in 100ml of alcohol.
Molisch’s test
To the Sample 2-3 drops of 1% alcoholic - napthol solution and 2 ml of conc. sulphuric acid was added
along the sides of the test tube. Appearance of purple to violet ring at the junction of two liquids shows
the presence of carbohydrates.
Anthrone test: 2 ml of anthrone reagent was added to 500 μl of extract. Formation of green blue colour
gives a positive anthrone test.
*Anthrone is a tricyclic aromatic ketone. It is used for a common cellulose assay and in the colorometric
determination of carbohydrates.
** Carbohydrate is first hydrolysed into simple sugars using dilute hydrochloric acid. In hot acidic
medium glucose is dehydrated to hydroxmethyl furfural. This compound forms with anthrone a green
coloured product with absorption maximum at 630 nm.
Test for Reducing Sugars:
Fehling test:
2ml solution of Fehling A and Fehling B were taken in a test tube then dropwise sample were added. The
mixture was shaken well and kept in a water bath for 10-15 minutes at 100 0C. A rusty brown or brick red
colour precipitate confirms the presence of carbohydrates in the sample.
Fehling solution:
a) Fehling's solution A (Copper sulfate solution): 3.46g of CUSO4. 5H2O was dissolved in 50ml of
distilled water.
b) Fehling's solution B (Alkaline tartrate solution): 17.3g of potassium sodium tartrate (Rochelle salts,
KNaC4H406. 4H2O) and 5g of NaOH were dissolved in 50 ml of distilled water.
Benedict test:
2ml of Benedict reagent was added to the 1 ml of plant extract. Then the mixture was shaken well and
placed in a water bath for 10-15 minutes. Formation of reddish precipitate indicates the presence of sugars
in the sample.

2. Benedict's solution (Fehling's solution) is used to test for simple sugars such as glucose. It is a clear
blue solution which is a combination of copper sulfate, sodium citrate, and sodium carbonate.
Test for Mono-saccharides
Barfoed’s Test: This reagent was prepared by dissolving 13.3 g of crystalline neutral copper acetate in
200 ml of 1 % acetic acid solution. The test residue dissolved in water and heated with a little of the
reagent. If a red precipitate of cuprous oxide is formed within two minutes, mono-saccharides are present.
Test for Starch: 1 ml of iodine solution is mixed in 1ml of extract; formation of blue colour indicated the
presence of starch in the extract.
Test for mucilage and gums
Small quantities of sample was added separately to 25 ml of absolute alcohol with constant stirring and
filtered. The precipitates was dried in oil and examined for its swelling property for the presence of gum
and mucilage.
To the sample add ruthenium red solution, pink color shows presence of mucilage.
Test for proteins and free amino acids:
Biuret test: 1% of NaoH was added to 1 ml of extract and few drops of 1% CuSO4 were then added.
Blue/ purple or violet/ pinkish colour indicates the presence of proteins.
*The biuret test, also known as Piotrowski's test.
**The Biuret reagent is made of sodium hydroxide (NaOH) and hydrated copper(II) sulfate, together with
potassium sodium tartrate,[5] the latter of which is added to chelate and thus stabilize the cupric ions. The
reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads to the displacement of
the peptide hydrogen atoms under the alkaline conditions. A tri- or tetra-dentate chelation with the peptide
nitrogen produces the characteristic color.
Millon’s test: 1 ml of test extract was mixed with H2SO4 then Millon’s reagent was added dropwise.
White/yellow precipitate appears which turns into brick red colour precipitate, after heating the mixture.
This indicates the presence of proteins and free amino acids.
*Millon's reagent is prepared by dissolving mercuric nitrate in nitric acid, and then adding water to dilute
it.
**Millon's reagent is an analytical reagent used to detect the presence of soluble proteins. A few drops of
the reagent are added to the test solution, which is then heated gently. A reddish-brown coloration or
precipitate indicates the presence of tyrosine residue which occur in nearly all proteins.
Ninhydrin’s test: 2 drops of freshly prepared Ninhydrin reagent (0.1% in n- butanol) is added to 1ml of
extract and heat and observed for blue or red orange colour.
* Ninhydrin (2,2-dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and
secondary amines. Ninhydrin is most commonly used to detect fingerprints, as the terminal amines of
lysine residues in peptides and proteins sloughed off in fingerprints react with ninhydrin. It is a white
solid which is soluble in ethanol and acetone at room temperature.

3. ** Ninhydrin reacts with the α-amino group of primary amino acids producing 'Ruhemann's purple'. The
chromophore formed is the same for all primary amino acids. The intensity of the colour formed depends
on the number and chemical nature of the amino groups being analysed. The optimum pH for the overall
reaction is 5.5.
Test for Xanthoproteins: One ml of extracts is treated separately with few drops of conc. HNO3 and
NH3 solution. Formation of reddish orange precipitate indicates the presence of xanthoproteins.
Tests for fixed oils and fats:
Spot test: A small quantity of sample was separately pressed between two filter papers. Appearance of oil
stain on the paper indicates the presence of fixed oil.
Saponification test: Few drops of 0.5 N alcoholic potassium hydroxide were added to a small quantity of
sample along with a drop of phenolpthlein, the mixture was heated on a water bath for 1-2 hours,
formation of soap or partial neutralization of alkali indicates the presence of fixed oils and fats.
*Saponification is the process by which the fatty acids in the triglycerides or fat are hyrdrolysed by an
alkali to give glycerol and potassium salts of fatty acids. A known quantity of fat or oil is refluxed with an
excess amount of alcoholic KOH. The value obtained is used for the determination of saponification
number.
Test for Alkaloids:
Mayer’s test: 1ml of sample was added to a few drops of Mayer’s reagent. Formation of white or pale
yellow precipitate indicates the presence of alkaloids in the sample.
* Mayer's reagent: 5g of KI was dissolved in 10ml of water and 1.35g of HgCl2 was dissolved in 60ml
of water. Then both the solutions were mixed together and water was added to make the solution to
100ml.
Wagner’s test: 1.5% of HCl was added in 1 ml of extract and a few drops of Wagner’s reagent were
added to it. Appearance of yellow/ brown precipitate indicates the presence of alkaloids. Hager’s test: 1ml
of extract was taken in a test tube, and few drops of Hagers reagent was added to it. Yellow precipitate
confirms the presence of alkaloids in the sample.
* Wagner's reagent (lodo-potasium iodide): 2g of iodine and 6g of KI was dissolved in 100ml of water
Dragendorff test: 5ml of distilled water was added to the the 2 ml of sample, then 2M HCl and 1 ml of
Dragondrof’s reagent (potassium bismuth iodide solution) was added. Orange / orange red precipitate
indicates the presence of alkaloids.
Hager’s Test: When the test filtrate was treated with this reagent, an orange yellow precipitate was
formed indicating the presence of alkaloids.
*Hager's reagent (Picric acid): 1g of picric acid was dissolve in 100ml of water to prepare Hager's
reagent (saturated aqueous solution of picric acid).
Valser’s test: A few drops of Valser’s reagent are added to a few mL of plant extract. A reddish-
brown precipitate indicates a positive result.

4. *Valser’s reagent is Potassium iodide solution in Mercuric iodide.
Tannic acid Test: A few drops of 10% Tannic acid solution are added to a few mL of plant extract.
A Buff coloured precipitate indicates a positive result.
Test for Glycosides
Legal’s test: To the sample add 1 ml of pyridine and few drops of sodium nitropruside solutions and then
it was made alkaline with sodium hydroxide solution. Appearance of pink to red colour shows the
presence of glycosides.
Borntrager’s test: Sample was treated with chloroform and then the chloroform layer was separated. To
this equal quantity of dilute ammonia solution was added. Ammonia layer acquires pink color, showing
the presence of glycosides.
Baljet’s test: To the sample add Sodium picrate solution, orange color shows presence of glycosides.
Test for cardiac glycosides:
Keller-Killani test: Treat the extract with 2 ml of glacial acetic acid containing one drop of ferric
chloride solution. Add 1ml of conc. sulphuric acid. Appearance of brown ring at the interface indicates
the deoxysugar characteristic of cardenolides. Appearance of a violet ring below the brown ring & a
greenish ring in the acetic acid layer confirmed the results.
Test for tannins and phenolic compounds
A small quantity of the sample was taken separately in water and test for the presence of phenol
compounds and tannins was carried out with the following reagents:
Lead acetate test: The extract (50 mg) is dissolved in distilled water. Then 3 mL of 10% lead acetate
solution is added. A bulky white precipitate indicates the presence of phenolic compounds.
Potassium Dichromate Test: If on an addition of a solution of potassium dichromate in test filtrate, dark
color is developed, tannins are present.
Test for Tannins: To 1 ml of the extract, 2ml of 5% FeCl3 is added which gives dark blue or greenish
black colour and a positive tannin test.
Test for Phlobatannins:
1ml of plant extract was treated with 1 ml of 1% HCl and heat. Red colour precipitate indicates the
presence of Phlobatannins in the sample.
Test for flavonoids:
Alkaline reagent test
To the test solution add few drops of magnesium hydroxide solution, intense yellow colour is formed
which turns to colourless on addition of few drops of dilute acid indicates presence of flavonoids.
Shinoda’s test
Small quantities of the sample was dissolved in alcohol, to them piece of magnesium followed by conc.
hydrochloric acid drop wise added and heated. Appearance of pink, crimson red, green to blue color
shows the presence of flavonoids.

5. Bate–Smith and Metcalf test: 0.5 mL of concentrated hydrochloric acid is added to the extract. It is
then warmed on a water bath for 15 minutes and is observed for an hour. A strong red or violet colour
means a positive result.
Lead acetate Test: Treat the extract with few drops of lead acetate solution. Formation of yellow
precipitate indicated the presence of flavonoids.
H2SO4 test: A fraction of the extract was taken and treated with concentrated H2SO4 and observed for
the formation of orange colour.
Ferric Chloride Test: Add a few drops of ferric chloride solution to the extract solution. Development of
intense green colour indicates the presence of flavonoids.
Test for Anthocyanin and Betacynin:
1 ml of plant extract was treated with 1 ml of 2N NaOH then heated. Formation of bluish –green colour
indicated the presence of Anthocynin while yellow colour indicated the presence of betacynin.
Tests for steroids and triterpenoids
Libermann-burchard test
Treat the sample with few drops of acetic anhydride, boil and cool. Then add conc. sulphuric acid from
the side of test tube, brown ring is formed at the junction two layers and upper layer turns green which
shows presence of steroids and formation of deep red colour indicates presence of phytosterols and
triterpenoid.
Salkowski test
Mix 2 ml of chloroform to extract solution carefully added conc. Sulphuric acid (3 ml) to form a layer,
red colour at lower layer indicates presence of steroids and formation of yellow coloured lower layer
indicates presence of triterpenoids.
Test for Terpinoids: To 1ml of plant extract, 2ml of chloroform and 3ml of conc. H2SO4 was added. A
reddish brown precipitate at their interface, confirmed the presence of terpenoids.
Test for waxes: To the test solution add alcoholic alkali solution, waxes get saponified.
Test for Resins: 1ml of ethanolic extract was dissolved in acetone and then 1 ml of distilled water is
added. Turbidity indicates the presence of resins.
One ml of extract was treated with few drops of acetic anhydride solution followed by one ml of conc.
H2SO4. Resins give colouration ranging from orange to yellow.
Test for Carboxylic acid: One ml of the various extracts was separately treated with a few ml of
sodium bicarbonate solution. Effervescence (due to liberation of carbon dioxide) indicates the presence of
carboxylic acid.
Test for Coumarins: 0.5 g of the moistened various extracts was taken in a test tube. The mouth
of the tube was covered with filter paper treated with 1 N NaOH solution. Test tube was placed
for few minutes in boiling water and then the filter paper was removed and examined under the
UV light for yellow fluorescence indicated the presence of coumarins.

6. *Coumarins are a family of benzopyrones (1,2-benzopyrones or 2H-1-benzopyran-2-ones) widely
distributed in the nature. They represent an important family of naturally occurring and/or synthetic
oxygen-containing heterocycles, bearing a typical benzopyrone framework.
Test for Quinones: One ml of each of the various extracts was treated separately with alcoholic
potassium hydroxide solution. Quinines give coloration ranging from red to blue.
* The quinones are a class of organic compounds that are formally "derived from aromatic compounds
[such as benzene or naphthalene] by conversion of an even number of –CH= groups into –C(=O)– groups
with any necessary rearrangement of double bonds", resulting in "a fully conjugated cyclic dione
structure".