Folin and Ciocalteu

The colorimetric method based on the reagents of Folin and Denis or of Folin and Ciocalteu has been generally preferred over other methods to determine total phenols in complex natural materials such as wines and fruits (1, 2, 3, 4, 5). This method is relatively simple, convenient, reliable, generally applicable, and it is accepted as an official analysis in several countries for total phenols in wines and a number of other products. Although it is a preferred method, it can be even better than is commonly recognized. 

The biuret reaction (M16), the Folin and Ciocalteu reagent (K22), ultraviolet adsorption (H2) and polarographic determinations (Fig. 25) have also been proposed (B2, H16). 

The concentrations of phenolic compounds were determinedby HPLC (Waters) using a method described previously by Nilvebrant et al. (21). The total concentration of phenols was also estimated by a spectrophotometric method (23) based on the Folin and Ciocalteu s reagent (Sigma). 

Folin and Ciocalteu s reagent diluted 1 1 with water immediately before use... 

A. Blue color with 20% sodium carbonate and phenol reagent of Folin and Ciocalteu... 

The stock solution of Folin and Ciocalteu s reagent is prepared as follows Dissolve 100 g of sodium tungstate, Na2W0i,2H20, and 25 g of sodium molybdate, Na2Mo04,2H20, in 800 ml of water in a 1,500-ml flask, add 50 ml of concentrated phosphoric acid and 100 ml of concentrated hydrochloric acid and reflux for ten hours. Cool, add 150 g of lithium sulphate, 50 ml of water and 4 to 6 drops of bromine and allow to stand for two hours. Boil for fifteen minutes to expel excess bromine, cool, filter and dilute to 1 litre with water. Store at a temperature below 4" and use within four months of preparation do not use if any trace of green colour is present. 

Colorimetric determination is based on Folin and Ciocalteu s phenol reagent which gives a brilliant blue colour with as little as 0 01 mg of the alkaloid. 

Measure 2 ml of the standard solution (prepared by dissolving 10 mg of crystalline tubocurarine chloride in water and making up to 100 ml) or 2 ml of the test solution (prepared by diluting the solution under examination to approximately the same concentration as the standard) into a 25-ml glass-stoppered measuring cylinder. Add 3 ml of Folin and Ciocalteu s phenol reagent (see p. 476) and adjust the volume to 25 ml with water. Add 2 ml of 20 per cent sodium carbonate solution, mix and heat a suitable volume (5 ml) in a test-tube placed in a water-bath for three minutes. Cool the reaction mixture. 

Folin, O. Ciocalteu, V. 1927. On tyrosine and tryptophane determinations in proteins. J. Biol. Chem 73 627-650. 

Price, M.L. and Butler, L.G., Rapid visual estimation and spectrophotometric determination of tannin content of sorghum grain, J. Agric. Food Chem., 25, 1268, 1277, 1977. Folin, O. and Ciocalteu, V., On tyrosine and tryptophan determinations in proteins, J. Biol. Chem., 27, 627, 1927. 

SINGLETON v L, ORTHOFER R and LAMUELA-RAVENTOS R M (1999) Analysis of total phenolics and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent, Meth Enzymol, 299, 152-78. 

Singleton, V. L., Orthofer, R., and Lamuela-Raventos, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. In "Methods in Enzymology, Oxidant and Antioxidants (Part A)", (L. Packer, Ed.), vol. 299 pp. 152-178. Academic Press, San Diego, CA. 

Materials required Sample solution in methanol, distilled water, 20% Na2C03, Folin-Ciocalteu s phenol reagent, water bath, graduated tube, cuvette, micro pipette (0.1 ml), pipette of 1,2 and 10 ml, test tubes of 20 ml, pure ferulic acid (Serva, Germany), Shimadzu UV 160 spectrophotometer. 

Procedure A 0.002 ml aliquot of the sample solution in methanol was taken and 7 ml distilled water plus 0.1 ml Folin-Ciocalteu s phenol reagent was added and after 3 min 0.2 ml of 20% Na2C03 was included. After boiling at 90 °C (exactly 5 min) samples were cooled at room temperature and were diluted with HzO to 10 ml volume. Only distilled water and reagents were used as a blank. The absorbance of total phenolics was measured at 660 nm spectrophotometrically (a Shimadzu UV 160 spectrophotometer) as per Feldman and Hanks (1968), with a sensitivity of 0.05 pig/g d.w. A standard curve was constructed with different concentrations of ferulic acid (Serva, Germany). Concentrations of ferulic acid varied from 0.33-80jig/ml (Table 1). 

The Folin-Ciocalteu assay is the most widely used method to determine the total content of food phenolics (Fleck and others 2008). Folin-Ciocalteu reagent is not specific and detects all phenolic groups found in extracts, including those found in extractable proteins. A disadvantage of this assay is the interference of reducing substances, such as ascorbic acid (Singleton and others 1999). The content of phenolics is expressed as gallic acid or catechin equivalents. 

Magalhaes LM, Segundo MA, Reis S, Lima JLFC and Rangel AOSS. 2006. Automatic method for the determination of Folin-Ciocalteu reducing capacity in food products. J Agric Food Chem 54(15) 5241-5246. 

Abstract In this study, a new natural adsorbent (sumae leaves) for removing Cu (II) ion from the aqueous solutions has been investigated. Leaves of sumae were obtained from Siirt, Tmkey. The tannins were extraeted with acetone water (70 30, v/v) mixture from the leaves of sumac. For the total tannin determination Folin-Ciocalteu method was used and tannin content was found 27%. In batch experiments, pH profile, adsorption time, adsorbent/hquid ratio, initial concentration of metal ions, adsorbent amoimt, particle size of adsorbent and temperature were performed to determine binding properties of adsorbent for the Cu(II) ions. The concentrations of the metal ions in solutions before and after adsorption were measured with an atomic absorption spectrophotometer. 

A calibration graph 3-15 ppm TA concentration range was constructed and the content of tannins in sumac leaves was determined by the Folin-Ciocalteu method. 

Leaves of Sumac were used for removal of ions in aqueous solution. Tannins were extracted from the leaves of sumac by extracting with 70% (v/v) acetone-water solution. For the total tarmin determination Folin-Ciocalteu method was used and tannin content was found 27%. Various adsorption parameters for the effective removal of Cu + ions by using sumac leaves as an adsorbent from aqueous solutions were studied and optimized. 

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