The Tea Association of the USA, Inc.

All samples are analyzed in duplicate along with a control sample (one that has been analyzed previously).

Materials:

Folin & Ciocalteu's Phenol Reagent (Sigma)	Catalog item F-9252
Gallic Acid (Fisher Scientific)	Catalog item A122-500
Sodium Carbonate (Aldrich)	Catalog item 22353-0
Spectrophotometer	Capable of accurate measurements at the wavelengths required. (A Beckman DU650 is an example of the type of unit), use with methacrylate cells having a 1cm pathlength.
Pipettes	of various sizes e.g. Brand Tech Dispensette III Bottle-top dispensers and Eppendorf adjustable vol. pipettes.
Test Tubes	- 16 X 100mm (Fisher # 14-961-29)

Reagent Preparation:

(Careful measurements for all liquid dilutions and transfer steps should be made to assure precision of results. In addition, be very precise about all times for reactions in samples and standards.)

Prepare a saturated Sodium Carbonate Solution by adding 50g Sodium Carbonate to 200ml Deionized (DI) water in a 600ml beaker. The solution is stirred and heated until the Sodium Carbonate is completely dissolved. The solution is filtered and stored at Room Temperature until crystals precipitate. A seed crystal may be added to induce precipitation. If this does not work, place the beaker in a refrigerator for about 1 hour or until crystals appear.

Preparation of Standards:

Use Class-A volumetric glassware for preparing the standards. Prepare a 1000ppm stock solution of Gallic Acid by weighing 1.0050g Gallic Acid (adjusted for 2% moisture this will give 1000ppm solution) and dissolving in 1L deionised distilled water (1 ml Ethanol and/or use of a sonicator will help in the dissolution). Store this Stock Solution in an amber glass container in a refrigerator and use it to prepare fresh 'working standards' each time the analysis is performed. Remember to bring a portion of the Stock Solution to Room Temperature before using in a pipette.

Working Standards are prepared as follows:

200ppm	20mls stock solution to 100mls with DI water in a vol. flask.
150ppm	15mls stock solution to 100mls with DI water in a vol. flask.
100ppm	10mls stock solution to 100ml with DI water in a vol. flask.
50ppm	5mls stock solution to 100mls with DI water in a vol. flask
25ppm	5mls stock solution to 200mls with DI water in a vol. flask

 

Sample Preparation Methods for Tea Varieties

Tea Leaf samples (Green and Black):

Approximately 210g boiling Deionized (DI) water is added to 2.00g ±.0.005 sample of loose tea leaf in a 300ml beaker. The extraction is allowed to continue, on the bench top, for 4 minutes with stirring each minute. (If the tea leaf is in a typical tea bag, open the bag and remove the 2 gram sample). After 4 minutes the leaf is filtered through a 5 inch diameter circle of teabag paper into a tared beaker. (Please note that a powder funnel is used for the filtration step). The tea filter paper containing the extracted tea leaf is squeezed between two 3.5 inch watch glasses to express the remaining extract from the leaf into the beaker. The beaker with the tea extract is placed on a balance and the net weight brought to 200grams.

Instant Tea Powders and Diet Tea Powders:

Add 0.4-0.6g of tea powder (weighed accurately) to approximately 200g boiling DI water in a tared beaker. After cooling, bring to 200grams net weight. The amount of powder required for the test may need to be adjusted (higher or lower) if the antioxidant strength of the powder is not within the acceptable range of the standards for analysis.

Iced Tea Powders containing sugar:

Add approximately 20g of powder (weighed accurately) to approximately 200g boiling DI water in a tared beaker. After cooling, bring to 200 grams net weight. Again, the sample size may need adjustment to bring the strength within the range of the standards.

Solids

The percent solids of the extracts are measured by accurately weighing a 15-20g sample of the extract (in duplicate) into moisture determination dishes. Evaporate the bulk of the water on a steam bath and dry overnight (16hrs) in a vacuum oven at 70°C. (Alternatively, if there is no sugar present in sample, 1hr in a vacuum oven at 100°C may be used.)

Folin-Ciocalteu Reagent

(The MSDS for the Folin-Ciocalteu Reagent should be read before performing this test. All waste produced should be handled as required by the hazardous chemicals & waste regulations of the Federal and local agencies. General lab safety practices should be followed, including the use of gloves, safety glasses, etc. Reagent handling is best performed in a fume hood.) The Folin reagent is sensitive to reducing compounds, in this case polyphenols, thereby producing a blue color upon reaction. This blue color is measured spectrophotometrically. The procedure described herein is used to measure the relative polyphenol content of tea extracts and tea solutions, using Gallic Acid as a standard. The tea extract or solution should be prepared to an appropriate concentration so that the Absorbance readings are within the range of the standards. A reagent blank should be prepared at the same time. The samples are diluted and reagents are added as specified in the procedure. Full reaction and color development requires ONE hour for completion. The Absorbance of each solution is measured at 725nm along with the reagent blank and all standards. A regression analysis is performed on the standards (Absorbance vs. Concentration) and the final results for each sample is determined from that regression curve.

Method:

See the diagram for a convenient placement of tubes in a holder rack.) Place a series of test tubes in Row 3 of the rack and label --- Blank, 25ppm std., 50ppm std., 100ppm std., etc. and then Final sample 1, Final sample 2, Final sample 3, etc. Place a series of test tubes in Row 1 directly behind the 'sample' tubes and label --- 1:10 dil. #1, 1:10 dil. #2, 1:10 dil. #3 etc.

Prepare the 1:10 dilutions by pipetting 0.5ml of each sample extract into the corresponding test tube in Row 1. Add 4.5ml DI to each and vortex. Pipette 0.5ml of DI water into the "blank test tube" Pipette 0.5ml of each "Working Standard" into the corresponding marked test tube in Row 3 Pipette 0.5ml from each '1:10 dil' test tube in Row 1 into the corresponding 'Final sample' test tube in Row 3. (There should now be 0.5 ml in each test tube in Row 3.)

Starting with the "blank test tube" add 4.5mls DI water, 0.2ml Folin Reagent, 0.5ml Saturated Sodium Carbonate to each test tube and mix on the vortex unit. (Each of these additions should be completed within 15 seconds and in the exact order described.) Finally, add 4.3mls DI water to each and invert each tube to mix (place gloved thumb over the test tube and invert, wipe thumb on damp paper towel and proceed to next sample). Repeat this procedure for all the test tubes in the rack.

Allow samples to sit for a minimum of 1 hour at room temperature. (Allowing the samples to sit for a longer time is acceptable, as long as each sample and all standards are treated exactly the same and the time does not exceed 3 hours). Read the Absorbance of each tube on a spectrophotometer at 725nm using a 1cm cell. If the spectrophotometer is not capable of reading at 725nm, a wavelength of 765nm is acceptable but use the same wavelength for all measurements.

(Depending upon waste disposal laws in the area, all test tubes and cells should be emptied into a single container clearly labeled for disposal. The tubes should also be rinsed and the rinse solution added to that container. Dispose of the waste properly.)

Calculations:

If the Spectrophotometer being used is capable of performing the regression analysis and also provides the concentration calculations, then all that is needed is to use the factor (X10) to correct for the dilution performed during the procedure. If the Spectrophotometer does not perform the regression analysis then the next step is: Perform a linear regression of the Absorbance data for the Gallic Acid standards vs. Gallic Acid concentration and use the resulting graph along with the Absorbance data for the diluted samples to calculate the concentration of polyphenols in ppm for each sample. Correct these results for the 10X dilution. This data is then handled in the following manner:

Conc. In ppm of polyphenols in final sol'n (A) = Absorbance ¸ the "x" coefficient from the regression analysis.

Conc. of polyphenols (ppm) in sample (B) = Concentration of (A) X 10 (i.e.correcting for dilution made during procedure)

To convert ppm polyphenols (B) to mg polyphenols per 2g sample (C), divide (B) by 5

To convert (C) to mg of flavonoids in 2 gram sample, multiply (C) X 0.84, (this is a correction factor for nonflavonoid material that is detected by the Folin Reagent)

The last calculation (in BOLD type) should be done by the analyst, but if the work is performed by an outside lab, it should be confirmed that the reported data is a result of this calculation in BOLD type. Also note that this final result (for leaf) is for 2 grams. For tea leaf serving sizes greater or less than 2 grams (e.g. 2.25g), multiply the above result by the factor of D/2, where D is the amount of tea leaf in a serving (e.g. 2.25 g).