12. Phlobatannin test: The occurrence of phlobatannin can be confirmed by the
deposition of red precipitate when the aqueous solution of tannin is mixed with
5 ml of 1% hydrochloric acid (Ezeonu and Ejikeme 2016).
21.7.2 Quantitative Tests
1. Hide-powder method: The aqueous solution of tannin is mixed with previously
dried chromated hide powder in vacuum for 24 h. over CaCl 2 and the mixture is
stirred for 1 h. at ambient temperature. The suspension forms and it is filtered
through sintered glass filter. The weight gain of the hide-powder after filtration
is expressed as a percentage of the weight of the starting material is referred to
the percentage of tannin in the sample (Guangcheng et al. 1991).
2. Stiasny's method: In the aqueous solution of tannin, 1 ml of 10 (M) HCl and 2 ml
of 37% formaldehyde are added. The reaction mixture is heated over reflux for
30 min and it is then filtered through sintered glass. The precipitate obtained, is
washed with hot water for several times and dried over CaCl 2 bed. The percentage yield of tannin is calculated with the starting material (Zhang et al. 2008).
3. Folin–Ciocalteau spectrophotometric method: The Folin-Ciocalteu reagent
(FCR) or Folin-Denis reagent, is named after Otto Folin, Vintilă Ciocâlteu,
and Willey Glover Denis. This reagent is prepared by dissolving 10 g of sodium
tungstate and 2.5 g of sodium molybdate in 70 ml of water, followed by the
addition of 5 ml of 85% phosphoric acid and 10 ml of concentrated
hydrochloric acid and 15 g of lithium sulphate, 5 ml of water, and 1 drop of
bromine (Peterson 1979). Polyphenols in plant extracts react with the reagent to
form a phosphotungstic-phosphomolybdenum complex, which gives blue
fluorescence under UV visible spectrophotometry (Schofield et al. 2001). The
maximum absorption of the chromophore depends on the alkaline solution and
the concentration of phenolic compounds. The maximum absorption is generally
obtained in a wave length of 760 nm (Blainski et al. 2013).
4. Turbidimetric method: This method is based upon the simple precipitation
reaction between Cu
2+ from copper acetate and tannin at 4.5 pH and the
absorbance is being monitored at around 470 nm (McDonald et al. 1996). The
precipitate is formed due to the presence of functional groups such as
ortho-dihydroxyphenyl and carboxyl groups in tannins, which act as mono or
bidentate ligands and forms chelates (Lima et al. 2012).
5. Ferrous tartrate method (Colorimetric method): This method is based on the
formation, in a buffered medium (pH 6.8) of a complex between ferrous tartrate
and tannins in the tea, which absorbs light at around 560 nm (Iwasa and Torii
1962). The ferrous tartrate method is more selective than the Folin–Ciocalteau
method, and is unaffected by the co-existence of reducing agents, such as
ascorbic acid (Y T Hung et al. 2010).
21 Tannins and Polyphenols Extracted from Natural Plants …
735
deposition of red precipitate when the aqueous solution of tannin is mixed with
5 ml of 1% hydrochloric acid (Ezeonu and Ejikeme 2016).
21.7.2 Quantitative Tests
1. Hide-powder method: The aqueous solution of tannin is mixed with previously
dried chromated hide powder in vacuum for 24 h. over CaCl 2 and the mixture is
stirred for 1 h. at ambient temperature. The suspension forms and it is filtered
through sintered glass filter. The weight gain of the hide-powder after filtration
is expressed as a percentage of the weight of the starting material is referred to
the percentage of tannin in the sample (Guangcheng et al. 1991).
2. Stiasny's method: In the aqueous solution of tannin, 1 ml of 10 (M) HCl and 2 ml
of 37% formaldehyde are added. The reaction mixture is heated over reflux for
30 min and it is then filtered through sintered glass. The precipitate obtained, is
washed with hot water for several times and dried over CaCl 2 bed. The percentage yield of tannin is calculated with the starting material (Zhang et al. 2008).
3. Folin–Ciocalteau spectrophotometric method: The Folin-Ciocalteu reagent
(FCR) or Folin-Denis reagent, is named after Otto Folin, Vintilă Ciocâlteu,
and Willey Glover Denis. This reagent is prepared by dissolving 10 g of sodium
tungstate and 2.5 g of sodium molybdate in 70 ml of water, followed by the
addition of 5 ml of 85% phosphoric acid and 10 ml of concentrated
hydrochloric acid and 15 g of lithium sulphate, 5 ml of water, and 1 drop of
bromine (Peterson 1979). Polyphenols in plant extracts react with the reagent to
form a phosphotungstic-phosphomolybdenum complex, which gives blue
fluorescence under UV visible spectrophotometry (Schofield et al. 2001). The
maximum absorption of the chromophore depends on the alkaline solution and
the concentration of phenolic compounds. The maximum absorption is generally
obtained in a wave length of 760 nm (Blainski et al. 2013).
4. Turbidimetric method: This method is based upon the simple precipitation
reaction between Cu
2+ from copper acetate and tannin at 4.5 pH and the
absorbance is being monitored at around 470 nm (McDonald et al. 1996). The
precipitate is formed due to the presence of functional groups such as
ortho-dihydroxyphenyl and carboxyl groups in tannins, which act as mono or
bidentate ligands and forms chelates (Lima et al. 2012).
5. Ferrous tartrate method (Colorimetric method): This method is based on the
formation, in a buffered medium (pH 6.8) of a complex between ferrous tartrate
and tannins in the tea, which absorbs light at around 560 nm (Iwasa and Torii
1962). The ferrous tartrate method is more selective than the Folin–Ciocalteau
method, and is unaffected by the co-existence of reducing agents, such as
ascorbic acid (Y T Hung et al. 2010).
21 Tannins and Polyphenols Extracted from Natural Plants …
735