5. AOACSPDSMethods-2018AwardsV3

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AOAC Official Method 2017.13 Total Phenolic Content in Extracts Folin-C (Folin and Ciocalteu) Colorimetric Method First Action 2017

performance could be compromised if formulations vary from the prescribed product. ( c ) Sodium carbonate .—Anhydrous (Cat. No 7527, Mallinckrodt, Phillipsburg, NJ, USA, or equivalent). ( d ) Sodium carbonate solution (20% Na 2 CO 3 ) .—Transfer 200 g sodium carbonate into a 1000 mL volumetric flask. Fill to volume with water and mix well. ( e ) Gallic acid .—Crystalline, ≥98% (Cat. No. G7384; Sigma- Aldrich, or equivalent). Store desiccated at room temperature. D. Preparation of Test Solutions ( a ) Preparation of calibration standard solutions .—Accurately weigh about 1.1 (±0.1) g gallic acid, and transfer into a 1000 mL volumetric flask (record weight to nearest 0.0001 g). Add about 750 mL water and sonicate to dissolve. Dilute to volume with water and mix well. Label as “Stock Standard Solution.” This standard may be used for up to 1 month when stored at 2–8°C. Prepare calibration standard solutions as shown in Table 2017.13B by pipetting the indicated amount of stock standard solution into the indicated size volume flask and diluting to volume with water. Approximate concentrations of gallic acid in each of the calibration standard solutions are shown in Table 2017.13B . ( b ) Preparation of sample test solution .—Accurately weigh about 100 mg test material and transfer into a 100 mL volumetric flask. Add about 75 mL water and sonicate in an ultrasonic bath containing about 3 cm water for up to 10 min until solids are dissolved. Dilute to volume with water and mix well. Quantitatively pipet 5.0 mL of this solution into a 100 mL volumetric flask. Dilute to volume with water and mix well. Weight of test material can be adjusted based on total phenolic content in order to achieve an absorbance within the calibration curve range. E. Determination ( a ) Colorimetric reaction .—Prepare a series of seven test tubes, each containing 15.00 mL water and 1 mL Folin-C phenol reagent. Into each test tube, add one of the following: ( 1 ) 1.00 mL sample test solution; ( 2 ) 1.00 mL calibration standard solution 1–5; or ( 3 ) 1.00 mL water. Mix contents of each test tube well and allow to sit for 6 min. Add 3.0 mL sodium carbonate solution to each tube and mix well. Place test tubes in heating block for 120 min. ( b ) Measurement .—In duplicate, transfer 1 mL of each reaction to a cuvette and measure the absorbance at 765 nm after zeroing the spectrophotometer with water. Measure calibration standard solutions first, then sample test solutions. Report mean of duplicate measurements. F. Calculations ( a ) Stock standard solution .—Calculate concentration of gallic acid in stock standard solution as follows: Table 2017.13B. Preparation of calibration standard solutions Calibration std soln Vol. stock std soln, mL Vol. flask, mL Approx. concn gallic acid, mg/L 1 1 25 40 2 2 25 80 3 3 25 120 4 4 25 160 5 5 25 200

(Applicable to the determination of total polyphenolic content in grape seed, grape skin, coffee, cocoa, and black tea extracts with >5% total phenolic content.) See Table 2017.13A for comparison of method performance to SMPR 2015.009. A. Principle Phenols are first extracted in water, then reacted with the Folin-C reagent (a complex mixture of heteropolyphosphotungstate- molybdate) in the presence of sodium carbonate to form a colored blue complex. The intensity of the blue color is proportional to the amount of reactive phenolic compounds in the sample. Phenolic content is determined by measuring the absorbance of the sample solution at 765 nm and comparing to a calibration curve using gallic acid as a standard. The method is able to quantify total polyphenolic content of about 5–100% (w/w) in the extracts. The method will also yield positive results for extracts containing ascorbic acid, amino acids, or sugars. B. Apparatus Note : Equivalent apparatus may be substituted. All volumetric pipets and volumetric flasks are Class A. ( a ) UV-Vis spectrophometer .—Capable of measuring absorbance at 765 nm in 1 cm quartz cuvettes. ( b ) Analytical balance .—Accurate to ±0.01 mg. ( c ) Test tubes .—25 × 150 mm Pyrex rimless (VWR Cat. No. 60820-320, Corning No. 9820). ( d ) Volumetric flasks .—50, 100, and 1000 mL. ( e ) Volumetric pipets .—1, 2, 3, 4, 5, and 15 mL. ( f ) Adjustable pipettor .—1–5 mL. ( g ) Bottles .—1, 3, and 15 mL with bottle top dispensers. ( h ) Vortex mixer . ( i ) Dry block heater .—Capable of maintaining 30 ± 2°C and holding 25 × 150 mm tubes. ( b )  Folin-C phenol reagent .—Cat. No. F9252 (Sigma-Aldrich, St. Louis, MO, USA; www.sigmaaldrich.com). Note : Formulations of Folin-C phenol reagent vary among manufacturers, and method Table 2017.13A. Comparison of SMPR 2015.009 and Folin-C SLV results SLV parameter Gallic acid equivalent (w/w) SLV result Analytical range 5–500 ppm 5–100% LOQ ≤5 ppm ND a Recovery 80–110% 91–104% for GA RSD r 3–5 ppm ≤9% ND >5 ppm ≤7% <7.5% a  ND = Not determined. ( j ) Ultrasonic bath . ( k ) Ice water bath . ( l ) Cuvettes .—Quartz, 1 cm. C. Reagents ( a ) Water .—HPLC grade.

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