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Demonstrate the extraction and qualitative and quantitative estimation of secondary metabolites
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Qualitative Analysis

 

  • Phenolics: The qualitative tests for detection of phenolics in plant samples are given below:

 

  • FeCl3 Test: To 1 ml of the filtered plant extract sample, add few drops of 10% ferric chloride solution. Green blue or violet colour appears which indicates the presence of phenolic compounds.

 

  • Flavonoids:  The qualitative tests for detection of flavonoids in plant samples are given below:

 

  • Shinoda Test: Crude extract was mixed with few fragments of magnesium ribbon and concentrated HCl was added drop wise. Pink scarlet colour appeared after few minutes which indicated the presence of flavonoids.

 

  • Sodium hydroxide Test:  To 1 ml of the plant extract, add few drops of dilute NaOH solution.An intense yellow colour is observed. It becomes colourless on addition of a few drops of dilute acid  which indicates the presence of flavonoids.

 

  • Lead acetate Test: To the plant aqueous extract, add 10% lead acetate solution .Yellow precipitate is formed which indicates the presence of flavonoids.

 

 

Mechanism of Folin Ciocalteu (FC) method:

 

  • The Folin-Ciocalteu (FC) reagent which is a mixture of tungstates and molybdates works on the mechanism of oxidation-reduction reaction.

 

  • This method strongly relies on the reduction of the mixture heteropolyphosphotungsates-molybdates by the phenolic compound which results in the formation of the blue colored chromogen.

 

  • The reaction forms a blue chromophore constituted by a phosphotungstic phosphomolybdenum complex where the maximum absorption of the chromophores depends on the alkaline solution and the concentration of phenolic compounds.

 

  • The phenolic compounds react with FC reagent only under basic conditions adjusted by sodium carbonate solution.

 

  • Under basic conditions it has been observed that the phenolic compound undergoes dissociation to form a phenolate anion which reduces the FC reagent i.e. the mixture of tungstates and molybdates rendering a blue coloured solution.

 

  • The colour intensity is measured by using a UV spectrophotometer. However, this reagent rapidly decomposes in alkaline solutions, which makes it necessary to use an enormous excess of the reagent to obtain a complete reaction. This excess can result in precipitates and high turbidity, making spectrophotometric analysis impossible.

 

  • To solve this problem, Folin and Ciocalteu included lithium salts in the reagent, which prevented the turbidity. The reaction generally provides accurate and specific data for several groups of phenolic compounds, because many compounds change colour differently due to differences in unit mass and reaction kinetics.

 

 

Mechanism of Aluminium chloride method:

 

  • The spectrophotometric assay based on aluminium complex formation is one of the most commonly used procedure for the so-called total flavonoid determination, as the content of these compounds is considered as an important parameter for evaluating food or medicinal plant samples.

 

  • The method is based on the nitration of any aromatic ring bearing a catechol group with its three or four positions unsubstituted or not sterically blocked.

 

 

Quantitative estimation by Spectrophotometer

 

Spectrophotometry is based on the measurement of the fraction of light of a given wavelength transmitted (T) or absorbed (A) as it passes through the solution contained in a transparent cell. The absorbance or the optical density of a molecule absorbing in the ultraviolet or visible region depends on the molecular structure and is given by Beer-Lambert Law:

The concentration of a solute is directly proportional to the absorbance of the solution.

Where Transmittance T is defined as

 

The sensitivity of the spectrophotometric method is dictated by the magnitude of molar absorptivity and the minimum absorbance that can be measured with a required degree of certainty.

 

  • Deviations from Beer’s Law: Generally all the coloured compounds or substances absorbed in the UV-Visible range follow Beer’s law up to a particular concentration. So, absorbance is linearly related to the path length. Linearity of the curve is not maintained in higher concentrations. Several factors are responsible for deviation:

 

  • Real deviations: It includes solute-solute, solute –solvent and hydrogen bonding which can affect the charge density of the analyte environment and hence its absorptivity.

 

  • Instrumental deviations: This may be due to polychromatic radiation, stray radiations, mismatched cells and instrument noise.

 

  • Chemical deviations: these deviations are due to chemical effects such as dissociations, associations, complex formation, polymerization, precipitations, temperature effects and photochemical reactions. Some of the examples are given below:

 

 

 

 

 

 

 

 

 

 

 

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