Phytochemical analysis

Overview

Phytochemical analysis of specimens are important in research. They are essential to determine the bioactive components of specimens and it’s amount.

In this article, we will be focusing on the quantitative analysis and qualitative analysis. We are laying emphasis on the basic phytochemical compounds.

Are you ready?, Sure, let’s go!

Qualitative test.

Qualitative analysis of specimens are performed to identify the bioactive component of a plant or animal part. In qualitative phytochemical analysis, it focuses on the identification of components of the explant under study or examination. It works with test, inference and confirmation of an element or compound.

Herbal plants
Medicinal plants

phytochemicals are secondary plant metabolites. They’re not involved in growth and development of the plant they are present in. They serve protective function in plants, coloration and others such as dormancy.

These secondary plant metabolites affect metabolic processes in animals when consumed. They are used to affect so many metabolic processes. They are used in modern medicine to produce drugs. traditionally, they were/ is still using herbs as tradomedical products.

The freshly prepared crude extract are preferred for qualitative tests on a plant under study. They will qualitatively tested for the presence of phytochemical constituents as reported below;

Test for Tannin according to Harbone, 1973


In this phytochemical analysis, the processes for testing tanin is as follows

  • Few drops of 30% ferric chloride solution is added to 2ml of the plant extract.
  • A greenish black colouration indicated the presence of tannin.

Test for saponin according to Harbone, 1973

The phytochemical analysis to test for saponin is as follows

  • Exactly 2ml of the plant extract will be added to 5ml of distilled water and was shaken vigorously.
  • A stable froth upon standing indicated the presence of saponin.

Test for Flavonoid according to Harbone, 1973

The qualitative phytochemical analysis of flavonoid is as follows

  • Few drops of 20% NaOH solution were added to 2ml of the plant extract.
  • A yellowish colouration indicated the presence of flavonoid.

Test for Cardiac glycoside

Qualitative phytochemical analysis is achieved as indicated below

  • Keller-kiliani test was performed to assess the presence of cardiac glycosides.
  • The crude dry powder of the plant extract is treated with 1 mL of Ferric chloride (FeCl3) reagent (mixture of 1 volume of 5% FeCl3 solution and 99 volumes of glacial acetic acid).
  • A few drops of concentrated sulphuric acid (H2SO4) is added to this solution.
  • Appearance of greenish blue color within minutes indicated the presence of cardiac glycosides

Test for Reducing Sugars accordingHarbone, 1973

Barfoed‘s Test: this phytochemical analysis is carried out to test for general presence of reducing sugar in the sample

  • In a sample bottle, 5ml of distilled water is added to 0.1g of the plant leaf extract.
  • The mixture is shaken vigorously and filtered.
  • To the filtrate is added equal volumes of fehlings solutions A and B and shaken vigorously.
  • A brick red precipitate indicated the presence of reducing sugars.

Quantitative Test For Phytochemical.

This test is carried out to ascertain the amount of a given phytochemical in a plant extract under study. The quantitative phytochemical analysis of selected group of phytochemicals are stated below. Let’s dive in!

Determination of Alkaloid by Harbone,1973

The quantitative phytochemical analysis of alkaloids are stated below

  • Exactly 2g of the powdered sample is weighed into a beaker
  • 40ml of 10% ethanolic acetic acid is added and covered to stand for 4hours
  • After which it is filtered using a whatman filter paper
  • The plant leaf extract is dried to a quarter of its original volume with the aid of a water bath.
  • Few drops of concentrated ammonium hydroxide were added to the extract until precipitation was completed
  • The entire solution is allowed to settle and the precipitate was collected by filtration using a filter paper.
  • The precipitate on the filter paper is dried in an oven at 600c
  • It is cooled in a desiccator and reweighed.

The percentage alkaloid was calculated as: ×100

Where,

W1=Weight of filter paper

W2=Weight of filter paper + residue (alkaloid)

Determination of tannin by Van Buren and Robinson, 1981

The method for ascertaining the quantitative phytochemical analysis of tanin is stipulated below

  • Exactly 2g of the powdered sample is weighed into 100ml beaker.
  • 50ml of distilled water is added and shaken for 1 hour in a mechanical shaker.
  • This is filtered into a 50ml volumetric flask.
  • Then, 5ml of the filtrate is pipetted out in a separate test tube and mixed with 3ml of 0.1m FeCl3 in 0.1 HCl and 0.008 potassium Ferric Cyanide.
  • The absorbance is measured in a spectrophotometer at 720nm wavelength, within 10minutes.
  • A blank sample is prepared, treated as the sample and the colour developed measured at the same wavelength.
  • A standard is prepared using tannic acid.
  • Tannin (mg/100ml) = Absorbance of sample concentration of standard, Absorbance of standard 1

Determination of flavonoid by Boham and Kocipa, 1994

This phytochemical analysis procedure measures the amount of flavonoids in an extract. It’s as follows

  • Exactly 2g of the plant sample is extracted repeatedly with 100ml of 80% aqueous methanol at room temperature.
  • The whole solution is filtered using a whatman filter paper.
  • The filtrate is later transferred into a crucible and evaporated to dryness over a water bath before weighing.
  • The percentage flavonoid is calculated as;

% flavonoid= × 100
Where;
W1= weight of empty crucible
W2= weight of crucible + sample after evaporation.

Determination of Saponin by Obadoni and Ochuko, (2001) method.

This method of phytochemical analysis is a little time consuming, but effective in determining the amount of saponin in an extract

  • Exactly 2g of the powdered sample is dispersed in 200ml of 20% ethanol.
  • The suspension is heated over a water bath for 4hours with continuous stirring at 55’C.
  • The mixture is filtered and the residue re-extracted with another 200ml of 20% ethanol.
  • The combined extract is reduced to 40ml over water bath at about 90’C.
  • The concentrated extract is transferred into a 250ml separation funnel and 20ml of diethyl ether is added and shaken vigorously.
  • The aqueous layer is recovered while the ether layer is discarded.
  • The purification process is repeated. 60ml of n-butanol is added.
  • The combined n-butanol extract is washed twice with 10ml of 5% aqueous sodium chloride.
  • The remaining solution is heated in a water bath to evaporate.
  • After evaporation, the sample is dried in the oven at 60’C to a constant weight.The saponin content is calculated in percentage as;

% Saponin= ×
Where;
W1 = weight of saponin (weight of beaker and content)
W2 = weight of beaker.

These process can be used to effectively determine the presence and amount of phytochemical is a sample.

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