Phytochemical Screening: Complete Guide to Plant Extract Analysis with Tests, Results, and Precautions

Table of Contents

• Introduction
• Requirements for Phytochemical Screening
• Test for Alkaloids
• Test for Phenolic compounds
• Test for Saponins
• Test for Flavonoids
• Test for Phytosterols 
• Test for Terpenoids 
• Summary of Phytochemicals, Screening Tests, and Positive Results
• Precautions during Phytochemical Screening
• References

Introduction

• Phytochemical screening is crucial for identifying the potential pharmacological properties of plants and even microorganisms.
• Characterization and evaluation of phytoconstituents support the investigation of therapeutic claims associated with medicinal plants.
• Advanced techniques like Liquid Chromatography (LC), High-Performance Liquid Chromatography (HPLC), and Gas Chromatography (GC) provide accurate qualitative and quantitative analysis of phytochemicals.
• However, these high-throughput methods are expensive and often not easily accessible in many settings.
• Conventional phytochemical tests offer a more practical alternative—they are simple, cost-effective, and require minimal resources, making them ideal for preliminary screening.

Requirements for Phytochemical Screening

Apparatus required

• 10 ml test tubes
• Measuring cylinders
• Micropipettes with compatible tips
• Sterile droppers

Chemicals Required

• Dilute hydrochloric acid (HCl)
• Potassium iodide (PI)
• Iodine solution
• Dilute sulphuric acid (H₂SO₄)
• Concentrated sulphuric acid (H₂SO₄)
• Lead acetate solution
• Chloroform
• Sodium hydroxide (NaOH)
• Mercuric chloride
• Ferric chloride solution
• Sodium bicarbonate
• Magnesium ribbon
• Glacial acetic acid
• Bismuth carbonate
• Picric acid solution
• Distilled water

Reagents/Solutions and their Composition

Mayer’s Reagent:

• Solution A: Dissolve 1.358 grams of mercuric chloride in 50 ml of distilled water.
• Solution B: Dissolve 5 grams of potassium iodide in 10 ml of distilled water.
• Working Solution: Mix Solution A and B, then add distilled water to make the final volume up to 100 ml.

Dragendorff’s Reagent:

• Prepare the stock solution by boiling 5.2 grams of bismuth carbonate, 4 grams of sodium iodide, and 50 ml of glacial acetic acid together for a few minutes.

Wagner’s Reagent:

• Dissolve 1 gram of iodine and 3 grams of potassium iodide in 50 ml of distilled water.

Hager’s Reagent:

• Dissolve 1 gram of picric acid in 100 ml of distilled water.

Test for Alkaloids

1. Mayer’s Test

Principle:

Alkaloids contain a nitrogen atom that can form a coordinate covalent bond with potassium ions in Mayer’s reagent, which is chemically known as potassium tetraiodomercurate. This interaction results in the formation of a precipitate.

Protocol:

• Add 1 ml of plant extract into a test tube.
• Add a few drops of Mayer’s reagent.
• Shake the mixture gently.

Positive Result:

Formation of a cream-colored, yellowish, or white precipitate indicates the presence of alkaloids.

2. Dragendorff’s Test

Principle:

Dragendorff’s reagent is composed of bismuth subnitrate and potassium iodide in acidic conditions (commonly glacial acetic acid). The low pH is crucial for the reaction.

• Bismuth ions react with potassium iodide to form black precipitates of bismuth iodide:

Bi³⁺ + 3 KI → BiI₃ + 3 K⁺

• Excess KI can convert BiI₃ into a soluble orange complex of potassium tetraiodobismuthate:

BiI₃ + KI → K[BiI₄]

• Alkaloids with tertiary amine groups form ammonium salts in acidic environments:

R₃N + HX → [R₃NH]⁺ + X⁻

• These ammonium salts react with tetraiodobismuthate ions to form an orange-red insoluble complex:

[R₃NH]⁺X⁻ + K[BiI₄] → [R₃NH]⁺[BiI₄]⁻ + KX

Protocol:

• Place 2 ml of plant extract in a test tube.
• Add 1 ml of Dragendorff’s reagent.

Positive Result:

• An orange-red precipitate confirms the presence of alkaloids.

3. Wagner’s Test

Principle:

Wagner’s reagent contains iodine and potassium iodide. The iodine reacts with iodide ions to form triiodide (I₃⁻), giving a brownish solution. Alkaloids form a complex precipitate with potassium ions through covalent bonding with the nitrogen atom in the alkaloid molecule.

Protocol:

• Add 0.5 ml of plant extract to a test tube.
• Dissolve it in 1 ml of 1% (v/v) hydrochloric acid.
• Add 3 drops of Wagner’s reagent.

Positive Result:

• A reddish-brown precipitate indicates the presence of basic alkaloids.

Test for Phenolic compounds

1. Ferric Chloride Test

Principle:

Phenolic compounds react with ferric ions (Fe³⁺), forming colored complexes. The resulting color—blue, green, or red—depends on the type of phenol present. The reaction can be represented as:

6 PhOH + Fe³⁺ → [Fe(OPh)₆]³⁻ + 6 H⁺

Protocol:

• Dissolve 1 ml of plant extract in 1 ml of sterile distilled water in a test tube.
• Add 3 drops of 1% (w/w) ferric chloride (FeCl₃) solution.

Positive Result:

A blue-black or violet coloration indicates the presence of polyphenols and tannins.

2. Lead Acetate Test

Protocol:

• Add the plant extract to a test tube.
• Add 3 ml of 10% lead acetate solution to the mixture.

Positive Result:

• Formation of a white precipitate indicates the presence of phenolic compounds.

Test for Saponins

1. Foam Test

Principle:

Saponins possess both polar (glycosyl) and non-polar (steroid or triterpenoid) groups, making them surface-active agents. When shaken with water, the polar ends align outward while the non-polar portions face inward, forming stable foam due to surface tension reduction.

Protocol:

• Dissolve 0.5 grams of dried plant extract in 2 ml of distilled water in a sterile test tube.
• Shake the mixture vigorously for 30 seconds.

Positive Result:

Persistent foam observed even after 30 minutes confirms the presence of saponins.

2. Hemolysis Test

Principle:

Saponins exhibit hemolytic activity by disrupting erythrocyte (red blood cell) membranes. They form insoluble complexes with cholesterol, leading to cell lysis and a visible zone of hemolysis.

Protocol:

• Place a few drops of fresh blood on a clean glass slide.
• Mix with the plant extract.

Positive Result:

A clear zone of hemolysis around the sample indicates the presence of saponins.

Test for Flavonoids

1. Alkaline reagent test

Principle: 

Flavonoids containing free hydroxyl groups at the C-2 position form stable anions in alkaline conditions, resulting in a yellow-colored solution when treated with sodium hydroxide (NaOH). Upon the addition of dilute hydrochloric acid (HCl), the medium becomes acidic, and the flavonoids revert to their original form, causing the yellow color to disappear.

Protocol:

• Take 2 ml of plant extract in a test tube.
• Add 2–3 drops of sodium hydroxide (NaOH).
• Then add 5 ml of dilute hydrochloric acid (HCl).

Positive Result:

An intense yellow color that disappears upon acidification indicates the presence of flavonoids.

2. Shinoda’s Test (Magnesium-Hydrochloride Reduction Test)

Principle:

In the presence of magnesium and hydrochloric acid, flavones (yellow) are reduced to anthocyanidins, resulting in a color change from orange to deep red. This indicates the presence of flavanol glycosides.

Protocol:

• Add a few milliliters of plant extract to a test tube.
• Add 10 drops of dilute hydrochloric acid (HCl).
• Introduce a small piece of magnesium ribbon.
• Add a few drops of concentrated HCl to dissolve the magnesium.

Positive Result:

The development of orange to deep-red coloration confirms the presence of flavonoids.

Test for Phytosterols 

1. Salkowski’s Test

Principle:

Salkowski’s test is based on a dehydration reaction of sterols in the presence of chloroform and concentrated sulfuric acid (H₂SO₄), which is highly hygroscopic. When sterols such as cholesterol are treated, water is removed from two molecules, forming two double bonds and resulting in a dimeric compound known as bisterol. In the case of cholesterol, bi-cholestadien forms and undergoes sulfonation, producing a red-colored complex.

Protocol:

• Add a few drops of concentrated sulfuric acid (H₂SO₄) to the plant extract.
• Shake the mixture well and allow it to stand.

Positive Result:

• The development of a red color indicates the presence of phytosterols.

2. Liebermann-Burchard Test

Principle:

This test detects cholesterol by reacting it with acetic anhydride and concentrated sulfuric acid, resulting in a series of color changes. The reaction initially produces a violet color, which gradually shifts to blue or green in the presence of phytosterols like cholesterol due to the formation of a conjugated diene system.

Protocol:

• Take 5 ml of aqueous plant extract in a test tube.
• Add 2 ml of acetic anhydride and then 2 ml of concentrated sulfuric acid.

Positive Result:

A gradual color change from violet to blue or green confirms the presence of phytosterols.

Test for Terpenoids 

1. Salkowski’s Test

Principle:

Chloroform is used to extract terpenoids from the plant material. Upon addition of concentrated sulfuric acid (H₂SO₄), a chemical reaction occurs at the interface, producing a characteristic reddish-brown color, indicating the presence of terpenoids.

Protocol:

• Take 5 ml of plant extract in a test tube.
• Add 2 ml of chloroform to the extract.
• Add 1–2 drops of concentrated sulfuric acid (H₂SO₄).
• Shake the mixture gently and allow it to stand.

Positive Result:

The appearance of a reddish-brown interface confirms the presence of terpenoids.

Summary of Phytochemicals, Screening Tests, and Positive Results

Alkaloids:

• Mayer’s Test: A creamy white or yellow precipitate indicates the presence of alkaloids.
• Dragendorff’s Test: An orange-red precipitate confirms the presence of alkaloids.
• Wagner’s Test: A brown or reddish precipitate indicates alkaloid presence.

Phenolic Compounds:

• Ferric Chloride Test: A blue or violet coloration indicates phenolics or tannins.
• Lead Acetate Test: Formation of a white precipitate confirms the presence of phenolic compounds.

Saponins:

• Foam Test: The persistence of foam after 30 minutes indicates the presence of saponins.
• Hemolysis Test: The appearance of a clear zone of hemolysis confirms the presence of saponins.

Flavonoids:

• Alkaline Reagent Test: An intense yellow coloration that becomes colorless after adding dilute acid indicates flavonoids.
• Shinoda’s Test: A deep red coloration confirms the presence of flavonoids (flavanol glycosides).

Phytosterols:

• Salkowski’s Test: A red coloration indicates the presence of phytosterols.
• Liebermann-Burchard Test: A color change from violet to blue-green confirms phytosterols such as cholesterol.

Terpenoids:

• Salkowski’s Test: The appearance of a reddish-brown interface indicates the presence of terpenoids.

Precautions during Phytochemical Screening

• Preliminary phytochemical tests are qualitative and provide only limited information. For accurate identification, follow-up with quantitative analytical techniques is recommended.
• The choice of extraction solvent is critical. Using an incompatible solvent may result in false-negative outcomes.
• Always wash hands thoroughly after handling chemical reagents, especially toxic ones like Mayer’s reagent.
• Dragendorff’s reagent is light-sensitive and should be stored in a dark container to prevent degradation.
• Handle ferric chloride with care, as it is highly corrosive.
• Lead acetate, being a heavy metal compound, must be disposed of safely following hazardous waste protocols.
• During the foam test for saponins, ensure that foam persistence is observed for at least 30 minutes to confirm a positive result.
• In the Shinoda test, the reaction between magnesium ribbon and concentrated sulfuric acid is exothermic and can release harmful fumes; handle with caution and avoid inhalation.
• Strong acids such as H₂SO₄ and HCl must be handled properly using acid-resistant gloves and goggles.
• Wear appropriate Personal Protective Equipment (PPE), including lab coat, gloves, and safety goggles, throughout the experiment.
• Conduct all tests in a well-ventilated area or under a fume hood to avoid inhaling toxic vapors.

References

• Chemistry Notes. (n.d.-b). Alkaloids – Introduction, six chemical tests, classification, and health benefits. Retrieved March 27, 2025, from https://chemistnotes.com/natural/alkaloids-introduction-chemical-test/
• Dubale, S., Kebebe, D., Zeynudin, A., Abdissa, N., & Suleman, S. (2023). Phytochemical screening and antimicrobial activity evaluation of selected medicinal plants in Ethiopia. Journal of Experimental Pharmacology, 15, 51–62. https://doi.org/10.2147/JEP.S379805
• ResearchGate. (2019, February 28). How does Salkowski’s test for steroid content work? Retrieved from https://www.researchgate.net/post/How-does-Salkowskis-Test-for-Steroid-content-work
• Shaikh, J. R., & Patil, M. (2020). Qualitative tests for preliminary phytochemical screening: An overview. International Journal of Chemical Studies, 8(2), 603–608. https://doi.org/10.22271/chemi.2020.v8.i2i.8834
• Sharma, T., Pandey, B., Shrestha, B. K., Koju, G. M., Thusa, R., & Karki, N. (2020). Phytochemical screening of medicinal plants and study of the effect of phytoconstituents on seed germination. Tribhuvan University Journal, 35(2), 1–11.
• Laboratoryinfo.com Team. (2022, March 30). Mayer’s test for alkaloids – Procedure, mechanism, results, uses. Retrieved from https://laboratoryinfo.com/mayers-test-for-alkaloids-procedure/
• Zohra, S. F., Meriem, B., & Samira, S. (2012). Phytochemical screening and identification of compounds from Mallow.