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Qualitative tests for preliminary phytochemical screening: An overview

Article · March 2020

DOI: 10.22271/chemi.2020.v8.i2i.8834

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 International Journal of Chemical Studies 2020; 8(2): 603-608

P-ISSN: 2349–8528
E-ISSN: 2321–4902
www.chemijournal.com Qualitative tests for preliminary phytochemical
IJCS 2020; 8(2): 603-608
© 2020 IJCS screening: An overview
Received: 14-01-2020
Accepted: 19-02-2020
Junaid R Shaikh and MK Patil
Junaid R Shaikh
M.V.Sc. Scholar, Veterinary
DOI: https://doi.org/10.22271/chemi.2020.v8.i2i.8834
Pharmacology and Toxicology,
College of Veterinary and Animal
Sciences, Udgir, Dist. Latur, Abstract
Maharashtra, India Medicinal plants have been used in the treatment of various diseases as they possess potential
pharmacological activities including antineoplastic, antimicrobial, antioxidant, anti-inflammatory,
MK Patil analgesics, anti-diabetic, anti-hypertensive, antidiarrheal and other activities. Phytoconstituents
Assistant Professor, Department individually or in the combination, determine the therapeutic value of a medicinal plant. Alkaloids,
of Veterinary Pharmacology and flavonoids, phenolics, tannins, saponins, steroids, glycosides, terpenes etc. are some of the important
Toxicology, College of Veterinary phytochemicals with diverse biological activities. The pharmacological activity of a plant can be
and Animal Sciences, Udgir, predicted by the identification of the phytochemicals. Currently, phytochemicals are determined by
Dist. Latur, Maharashtra, India various modern techniques, but the conventional qualitative tests are still popular for the preliminary
phytochemical screening of plants.

Keywords: Medicinal plants, phytoconstituents, phytochemical screening, qualitative tests

Introduction
Phytochemicals (Greek: phyton = plant) are chemical compounds naturally present in the
plants attributing to positive or negative health effects [1]. Medicinal plants used in different
diseases and ailments are the richest bio reservoirs of various phytochemicals. The medicinal
properties of the plants are determined by the phytochemical constituents [2]. Some of the
important phytochemicals include alkaloids, flavonoids, phenolics, tannins, saponins, steroids,
glycosides, terpenes, etc. which are distributed in various parts of the plants [3]. Nature is a
unique source of structures of high phytochemical diversity representing phenolics (45%),
terpenoids and steroids (27%) and alkaloids (18%) as major groups of phytochemicals [4].
Although, these compounds seem to be non-essential to the plant producing them, they play a
vital role in survival by mediation of ecological interactions with competitors, protect them
from diseases, pollution, stress, UV rays and also contribute for colour, aroma and flavour
with respect to the plant. The metabolites produced by the plants to protect themselves against
biotic and abiotic stresses have turned into medicines that people can use to treat various
diseases [5,6].
Phytochemicals can be separated from the plant material by various extraction techniques. The
most commonly used conventional methods include maceration, percolation, infusion,
digestion, decoction, hot continuous extraction (Soxhlet extraction) etc., recently, eco-friendly
techniques such as Ultrasound-Assisted Extraction (UAE), Microwave-Assisted Extraction
(MAE), Supercritical Fluid Extractions (SFE) and Accelerated Solvent Extraction (ASE) have
also been introduced [10,11]. Different types of solvents viz. water, ethanol, methanol, acetone,
ether, benzene, chloroform etc. are used in the extraction process [12]. Extraction of
phytochemicals from the plant materials is affected by pre-extraction factors (plant part used,
its origin and particle size, moisture content, method of drying, degree of processing etc.) and
extraction-related factors (extraction method adopted, solvent chosen, solvent to sample ratio,
pH and temperature of the solvent, and length of extraction) [10, 12].
Corresponding Author: Previously, the plant parts were directly used as such for the treatment, but now-a-days, the
Junaid R Shaikh active principles are identified and isolated in pure form and also synthetically produced with
M.V.Sc. Scholar, Veterinary
Pharmacology and Toxicology,
the help of advance techniques [6]. In the development of new synthetic drugs, the chemical
College of Veterinary and Animal structures derived from these phytoconstituents can be utilized as models [7]. Identification of
Sciences, Udgir, Dist. Latur, phytoconstituents in the plant material helps to predict the potential pharmacological activity
Maharashtra, India of that plant [8].
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Characterization and evaluation of plants and their unavailable or unaffordable, the conventional phytochemical
phytoconstituents can explore the evidences to support tests which are economic, easy and require fewer resources,
therapeutic claims of those plants against various ailments [12]. remain the good choice for preliminary phytochemical
Advanced techniques like Gas Chromatography (GC), Liquid screening [2]. The present communication deals with the
Chromatography (LC), High-Performance Liquid collection and compilation of maximum possible qualitative
Chromatography (HPLC), High-Performance Thin Layer phytochemical tests from various published literatures. The
Chromatography (HPTLC) etc. are very helpful for detection preliminary qualitative phytochemical tests for the detection
of phytoconstituents both qualitatively as well as of different phytoconstituents have been summarized in table
quantitatively [1]. However, when these techniques are 2.

Table 1: Reagent Preparation for Phytochemical Screening

Reagents/Solutions Composition
Stock solution: 5.2gm Bismuth carbonate + 4gm sodium iodide + 50mL glacial acetic acid, boiled for few min., After
12 hr. precipitated sodium acetate crystals are filtered by sintered glass funnel; 40mL filtrate + 160mL ethyl acetate +
1. Dragendroff’s reagent
1mL distilled water, (stored in amber-coloured glass bottle).
Working solution: 10mL stock solution + 20mL acetic acid + distilled water to make final volume 100mL.
2. Hager’s reagent Saturated aqueous solution of picric acid
Solution A : 1.358gm mercuric chloride + 60mL distilled water
3. Mayer’s reagent Solution B : 5gm potassium iodide + 10mL distilled water
Working solution: solution A + solution B + distilled water to make final volume 100mL
4. Wagner’s reagent 1.27gm iodine + 2gm potassium iodide + distilled water to make final volume 100mL
5. Barfoed’s reagent 30.5gm copper acetate + 1.8mL glacial acetic acid
6. Seliwanoff’s reagent 0.05 resorcinol + 100mL dilute HCl
Solution A: 173gm sodium citrate + 100gm sodium carbonate + 800mL water, dissolve & boil to make solution clear
7. Benedict’s reagent Solution B: 17.3gm of copper sulphate dissolved in 100mL distilled water
Working solution: Mix solution A and solution B
Solution A: 34.66gm copper sulphate + distilled water to make final volume 100mL.
8. Fehling’s solutions
Solution B: 173gm potassium sodium tartarate + 50gm NaOH + distilled water to make 100mL.
9. Baljet's reagent 95mL 1% picric acid + 5mL 10% NaOH
10. Millon’s reagent 1gm mercury + 9mL fuming nitric acid + equal amount of distilled water (after completion of reaction)

Table 2: Qualitative Tests for Phytochemical Screening

Observations
Test Procedure References
(Indicating Positive Test)
Detection of alkaloids
Dragendroff’s/ Kraut’s
1) Few mL filtrate + 1-2 mL Dragendorff’s reagents
a A reddish-brown precipitate [1, 21]
test
2) Hager’s test Few mL filtratea + 1-2 mL Hager’s reagents A creamy white precipitate [1, 7]

Mayer’s/ Bertrand’s/ Few mL filtratea + 1-2 drops of Mayer’s reagent (Along the [7, 12, 13]
3) A creamy white/yellow precipitate
Valser’s test sides of test tube)
Wagner’s test Few mL filtratea + 1-2 drops of Wagner’s reagent (Along [7, 21]
4) A brown/reddish precipitate
the sides of test tube)
5) Picric acid test Few mL filtratea + 3-4 drops of 2% picric acid solution An orange coloure [14, 15]

A blue colour, which disappears on boiling [16, 17]

6) Iodine Test 3mL extract solution + few drops of iodine solution
and reappears on cooling
6mL plant extract, evaporated completely + 6mL ethanol
7) Bouchardat’s test (@60 °C) + few drops of Bouchardat’s reagent (dilute A reddish brown colour [18]

iodine solution)
[30, 38]
8) Tannic acid test Acidified extract + 10% tannic acid solution A buff colour precipitate
Detection of Carbohydrates
1) Barfoed’s test 1mL filtrateb + 1mL Barfoed’s reagent + Heated for 2 min. A red precipitate {monosaccharides} [7, 19]

2mL filtrateb + 2 drops of alcoholic α–naphthol + 1mL conc.

2) Molish’s test A violet ring [7, 21]
H2SO4 (along the sides of test tube)
1mL extract solution + 3mL seliwanoff’s reagent + heated
3) Seliwanoff’s Test A rose red colour {ketoses} [17, 19]
on water bath for 1 min.
2mL aq. extract solution + few crystals of resorcinol + equal [13, 9]
4) Resorcinol test A rose colour {ketones}
volume of conc. HCl + heated
2mL conc. HCl + little amount of phloroglucinol + equal [13]
5) Test for pentoses A red colour
amount of aqueous extract solution + heated over flame
[20]
6) Test for starch Aqueous extract + 5mL 5% KOH solution A cinary colouration
Detection of Reducing sugars
0.5mL filtrateb + 0.5mL Benedict’s reagent + Boiled for 2
1) Benedict’s test Green/yellow/red colour [7, 21]
min.
1mL each of Fehling’s solution A & B + 1mL filtrateb +
2) Fehling’s test A red precipitate [7, 21]
boiled in water bath
Detection of Glycosides
Borntrager’s test 2mL filtrated hydrolysatec + 3mL Chloroform + shaken well [7]
1) A pink coloured solution
+ chloroform layer is separated + 10% Ammonia solution

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Plant extract + ferric chloride solution + boil for 5min. +

Modified Borntrager’s
2) cooled + equal volome of benzene + benzene layer is A rose-pink to blood red coloured solution [12, 33]
test
separated + Ammonia solution
Dissolve 50gm plant extract in pyridine +
3) Legal’s test A pink coloured solution [7, 12]
Sodium nitroprusside + 10% Sodium hydroxide
1mL dil. H2SO4 + 0.2mL extract + boiled for 15min. +
4) 10% NaOH test allowed cooling + neutralize with 10% NaOH + 0.2mL A brick red precipitate [21]
Fehling’s solution A & B
Alcoholic extract + dissolved in 1mL of water + few drops
5) Aqueous NaOH test A yellow colour [22]
of aqueous NaOH solution
5ml plant extract + 2mL glacial acetic acid + a drop of 5%
6) Concentrate H2SO4 test A brown ring [3]
FeCL3 + conc. H2SO4
7) Raymond’s test Extract solution + dinitrobenzene in hot methanolic alkali A violet colour [38]
Detection of Cardiac Glycosides
1mL filtrate + 1.5mL glacial acetic acid +
A blue coloured solution [21, 38]
1) Keller-Killani test 1 drop of 5% ferric chloride + conc. H2SO4 (along the side
(in acetic acid layer)
of test tube)
4mL extract evaporated to dryness + 1-2 mL methanol + 1-2
A disappearing violet colour
2) Kedee’s test mL alcoholic KOH + 3-4 drops of 1% alcoholic 3,5- [22]
{Cardenolides}
dinitrobenzene + heated
[20]
3) Test for Cardenolides Extract + pyridine + Sodium nitroprusside + 20% NaOH A red colour, fades to brownish yellow
[30]
4) Bromine water test Plant extract + few mL of bromine water A yellow precipitate
5) Baljet test 2mL extract + a drop of Baljet’s reagent A yellow-orange colour [39, 40]

Detection of Proteins and Amino acids

2mL filtrate + 1 drop of 2% copper sulphate sol. + 1mL of A pink coloured sol. [1, 7]
1) Biuret test
95% ethanol + KOH pellets (in ethanolic layer)
2) Millon’s test 2mL filtrate + few drops of Millon’s reagent A white precipitate [1, 7]

2mL filtrate + 2 drops of Ninhydrin solution (10mg A purple coloured sol. [1, 7]
3) Ninhydrin test
ninhydrin + 200mL acetone) {Amino acids}
[1, 12]
4) Xanthoproteic test Plant extract + Few drops of conc. Nitric acid A yellow coloured sol.
Detection of Flavonoids
1mL extract + 2mL of 2% NaOH solution An intense yellow colour, becomes [20, 21, 23]
1) Alkaline reagent test (+ few drops dil. HCl) colourless on addition of diluted acid
[7]
Plant extract + 10% ammonium hydroxide sol. A yellow fluorescence
[1, 21, 12]
2) Lead acetate test 1mL plant extract + few drops of 10% lead acetate solution A yellow precipitate
Shinoda’s test/ Mg- Plant extract is dissolved in 5mL alcohol +
A pink to crimson coloured solution [7, 38]
3) hydrochloride Fragments of magnesium ribbon +
{flavonal glycosides}
reduction test few drops of conc. HCl
Shibata’s reaction/ 1gm Aq. extract + dissolved in 1-2 mL 50% methanol by A red colour {flavonols}, orange colour [13, 22]
4)
Cyanidin test heating + metal magnesium + 5-6 drops of conc. HCl {flavones}
Extract aqueous solution + few drops 10% ferric chloride [20]
5) Ferric chloride test A green precipitate
solution
Few mL aqueous extract solution + 0.1gm metallic zinc +
6) Pew’s test A red colour {flavonols} [13]
8mL conc. H2SO4
Zinc-hydrochloride Plant extract + pinch of zinc dust + conc. HCl along the side [24, 25]
7) Magenta colour
reduction test of test tube
[26]
8) Ammonia test Filtrate + 5mL dil. Ammonia solution + conc. H2SO4 A yellow colour
[35]
9) Conc. H2SO4 test Plant extract + conc. H2SO4 An orange colour
Detection of Phenolic compounds
[21]
1) Iodine test 1mL extract + few drops of dil. Iodine sol. A transient red colour
[7, 12]
2) Ferric chloride test Extract aqueous solution + few drops 5% ferric chloride sol. Dark green/bluish black colour
Plant extract is dissolved in 5mL distilled water + 1% [7]
3) Gelatin test A white precipitate
gelatin solution + 10% NaCl
Plant extract is dissolved in 5mL distilled water + 3mL of [7]
4) Lead acetate test A white precipitate
10% lead acetate sol.
Plant extract aqueous solution + 5% glacial acetic acid + 5% Solution turns muddy / [3, 16]
5) Ellagic Acid Test
sodium nitrite solution Niger brown precipitate
Potassium dichromate [27]
6) Plant extract + few drops of potassium dichromate solution A dark colour
test
Warm water in beaker + mature plant part is dipped + Black or brown colour ring at the junction [34]
7) Hot water test
warmed for a min. of dipping
(1gm extract + 10mLchloroform, vigorously shaken and [35]
8) Test for Cartenoids A blue colour at the interface
filtered). Filtrate + conc. H2SO4
Detection of Tannins
Plant extract is dissolved in 5mL distilled water + 1% [12, 33]
1) Gelatin test A white precipitate
gelatin solution + 10% NaCl
d
1mL filtrate + 3mL distilled water + 3 drops 10% Ferric
2) Braymer’s test Blue-green colour [21, 28]
chloride solution
Formation of emulsion [21]
3) 10% NaOH test 0.4mL plant extract + 4mL 10% NaOH + shaken well
{Hydrolysable tannins}

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4) Bromine water test 10 ml of bromine water + 0.5gm plant extract Decoloration of bromine [23]

5) Lead sub acetate test 1mL filtratee + 3 drops of lead sub acetate solution A creamy gelatinous precipitate [3]

(5mL aq. extract + 0.5 g of sodium acid phosphate, heated,

[9]
6) Phenazone test allowed to cool + filtered); filtrate + 2% solution of Precipitation
phenazone
A water-soluble iron-tannin complex,
Extract solution + iron + sodium tartarate
7) Mitchell’s test which is insoluble in solution of [39]
(+ ammonium acetate solution)
ammonium acetate
Detection of Phlobatannins
[5, 13]
1) HCl test 2mL aq. extract + 2mL 1% HCl (boiled) A red precipitate
Detection of Saponins
[12]
0.5gm plant extract + 2mL water (vigorously shaken) Persistent foam for 10 min.
20mL water in measuring cylinder + 50gm extract [7]
Formation of 2cm thick layer of foam
1) Foam test (vigorously shaken for 15 min.)
0.2gm plant extract + 5mL distilled water; shaken well; Appearance of creamy miss of small [29]
heated to boiling bubbles
Plant extract + few mL sodium bicarbonate solution + [30]
2) NaHCO3 test Stable honeycomb like froth
distilled water (vigorously shaken)
Aq. extract + 5mL distilled water; shaken vigorously + few [23, 26]
3) Olive oil test Appearance of foam
drops of olive oil + shaken vigorously
[3, 16]
4) Haemolysis test Drop of fresh blood on glass slide + plant extract Zone of hemolysis
Detection of Phytosterols
Filtratef + few drops of conc. H2SO4 Red colour
1) Salkowski’s test [21, 12]
(Shaken well and allowed to stand) (in lower layer)
Libermann-Burchard’s 50gm extract is dissolved in 2mL acetic anhydride + 1-2 [7]
2) An array of colour change
test drops of conc. H2SO4 (along the side of test tube)
0.5mL plant extract + 2mL of acetic anhydride + 2mL conc. [26]
3) Acetic anhydride test Change in colour from violet to blue/green
H2SO4
Pink ring / Red colour (in lower [23, 40]
4) Hesse's response 5mL aq. extract + 2mL chloroform + 2mL conc. H2SO4
chloroform layer)
[34]
5) Sulphur test Extract solution + pinch of sulphur powder Sulphur sinks to the bottom
Detection of Cholesterol
2mL extract + 2mL chloroform + 10 drops of acetic [22]
1) A red-rose colour
anhydride + 2-3 drops of conc. H2SO4
Detection of Terpinoides
2ml chloroform + 5mL plant extract, (evaporated on water [23]
1) A grey coloured solution
bath) + 3mL conc. H2SO4 (boiled on water bath)
Detection of Triterpinoides
Filtratef + few drops of conc. H2SO4 Golden yellow layer
1) Salkowski’s test [21]
(Shaken well and allowed to stand) (at the bottom)
Detection of Diterpenes
Plant extract is dissolved in distilled water + 3-4 drops of [12, 33]
1) Copper acetate test Emerald green colour
copper acetate solution
Detection of Lignins
[16, 38]
1) Labat test Extract solution + gallic acid A olive green colour
[16, 38]
2) Furfuraldehyde test Extract solution + 2% furfuraldehyde solution A red colour
Detection of Carotenoids
10mL extract evaporated to dryness + 2-3 drops of saturated A blue-green colour eventually changing to [22]
1) Carr-Price reaction
solution of antimony trichloride in chloroform red
Detection of Quinones
[21, 26]
1) Alcoholic KOH test 1mL plant extract + few mL alcoholic potassium hydroxide Red to blue colour
[17]
2) Conc. HCl test Plant extract + conc. HCl A green colour
10mg extract + dissolved in isopropyl alcohol + a drop of [32]
3) Sulphuric acid test A red colour
conc. H2SO4
Detection of Anthraquinones
10mL 10% ammonia sol. + few ml filtrateg (shaken
1) Borntrager’s test A pink, violet, or red coloured solution [5, 23, 28]
vigorously for 30 sec.)
Ammonium hydroxide 10mg extract is dissolved in isopropyl alcohol + a drop of [32]
2) Formation of red colour after 2 min.
test conc. ammonium hydroxide solution
Detection of Anthocyanins
2mL plant extract + 2mL 2N HCl Pink-red sol. which turns blue-violet after [18, 31]
1) HCl test
(+ Few mL ammonia) addition of ammonia
Detection of Leuconthocyanins
[31]
1) Isoamyl alcohol test 5mL plant extract + 5mL isoamyl alcohol Upper layer appears red
Detection of Carboxylic acid
[21, 26]
1) Effervescence test 1mL plant extract + 1mL sodium bicarbonate solution Appearance of Effervescence
Detection of Coumarins
0.5gm moistened extract is taken in test tube, mouth of test
Yellow fluorescence from paper under the [21, 26]
1) NaOH paper test tube is covered with 1N NaOH treated filter paper, heated
UV light
for few min. in water bath
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[27]
2) NaOH test Plant extract + 10% NaOH + Chloroform A yellow colour
Detection of Emodins
[29]
1) Plant extract + 2mL NH4OH + 3mL benzene A red colour
Detection of Gums and Mucilages
Dissolve 100mg extract in 10mL distilled water + 25mL [7]
1) Alcohol test White or cloudy precipitate
absolute alcohol (constant stirring)
Detection of Resins
1mL plant extract + Acetic anhydride solution + 1mL conc. [21, 26]
1) Acetic anhydride test Orange to yellow
H2SO4
1mL plant extract dissolved in acetone, poured in distilled [34]
2) Turbidity test water Turbidity
[36]
10mL extract + 20mL 4% HCl
Detection of Fixed Oils and Fat
Little quantity of plant extract is pressed in between to filter [7, 38]
1) Spot test/ Stain test Oil stain on the paper
papers
Extract + few drops of 0.5N alcoholic KOH + A drop of Soap formation or partial neutralization of [7,[38]
2) Saponification test
phenolphthalein (Heated for 2hr.) alkali
3) Extract solution is applied on filter paper A transparent appearance {oils and resins} [35, 36]
Detection of Volatile Oils
[37]
1) Fluorescence test l0 mL of extract, filtered till saturation, exposed to UV light Bright pinkish fluorescence
a = 50gm solvent free extract is mixed with few mL dil. HCl and then filtered
b = 100mg solvent free extract is dissolved in 5mL of distilled water and filtered
c = 50gm of plant extract is hydrolysed with conc. HCl for 2 hr on water bath and filtered
d = 3gm powdered sample boiled in 50mL distilled water for 3 min. and then filtered
e = Small quantity of extract boiled with 5mL of 45% ethanol for 5 min. them cooled and filtered
f = Equal quantity of chloroform is treated with plant extract and filtered
g = 3mL of aq. extract is shaken with 3mL of benzene and filtered [5] OR 10mL of benzene is added in the plant sample and soaked for 10 min.

then filtered [23] OR extract is macerated with ether and filtered [28].
{ }=Indicates presence of specific phytoconstituents.
Note: The compositions of various reagents and solutions denoted by italic font have been mentioned in table 1.

Conclusion 7. Raaman N. Phytochemical Techniques. New India

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