1. Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate
Zhang Hui
J. Agric. Food Chem.2002,50,

2. Contents Introduction Materials and methods Results and discussion
Conclusion

3. Introduction Tannins gallotannins hydrolyzable tannins
condensed tannins
high molecular weight polyphenolics found in higher plants
gallotannins
ellagitannins

4. Structural formulas of representative hydrolyzable tannins from plants
Structural formulas of representative hydrolyzable tannins from plants. Compound 1 is pentagalloyl glucose, comprised of the gallate (G,2) esters of glucose. Compound 3 is the simple ellagitannin eugeniin, comprised of both the gallate esters and a single HHDP (G−G,4) ester. Compound 5 is EGCG, the gallate ester of the flavanoid epigallocatechin. Compound 6 is oenothein B, a macrocyclic hydrolyzable tannin. All four of thehydrolyzable tannins react in acidic methanol to yield methyl gallate (7)

5. Introduction
Individual hydrolyzable tannins can usually be determined by HPLC, but methods for conveniently screening large numbers of samples with variable hydrolyzable tannin compositions have not been developed.
The KIO3 method has been employed in some studies of oaks and maples.
The purpose of this work was to optimize the KIO3 method for analysis of hydrolyzable tannins.

6. Materials and methods HPLC Analysis of Methanolysis Products Reagents
tannin acid，gallic acid, methyl gallate, epigallocatechin gallate (EGCG)
Methanolysis and pH Adjustment
anhydrous methanol, 85 ℃, 20h, pH: 5.5±0.1
Analysis
30 ℃ water bath，
50 min after KIO3 was added, recorded absorbance at 525 nm
HPLC Analysis of Methanolysis Products
C18 (100×4.6 mm),1.0 mL/min , ƛmax 525 nm,
gradient elution

7. Results and Discussion1 2
+ KIO3 A B
ƛmax nm
yellow
Why KIO3 ？
Hypochlorite ?
the lifetime of A was shorter
Potassium periodate (KIO4) ?
past the red intermediate to the final product

8. Improve Disadvantages of KIO3 method different color yields
in rates of reaction
for structurally
different gallotannins
Improve
Why not gallic acid ？
Methyl gallate 1 2
+ KIO3 A B
ƛmax nm
yellow

9. Methanolysis Why 85℃? avoid the disappearance of some methyl gallate
formation of unidentified phenolic products observed at higher temperatures
Why anhydrous methanol?
traces of water caused production of a mixture of gallic acid and methyl gallate

10. How to control pH? KIO3 Reaction too acidic, the chromophore A
did not accumulate
too basic, oxidation was accelerated
and only the yellow end product
Reaction of KIO3 with methyl gallate as a function of pH. Methyl gallate solutions dissolved in methanol were diluted with a series of solutions comprised of ethanolamine and ammonium acetate buffer prepared at various pH values between 4.5 and 7.0.

11. KIO3 Reaction product A was temperature-dependent and time-dependent
How to control temperature?
at room temperature, there were significant day-to-day variations
30 ℃ water bath
How to control time?
50 min after KIO3 was added, recorded absorbance at 525 nm

12. a linear relationship between methyl gallate and absorbance at 525 nm:
Abs = μg
where R2 = 0.996, standard error of Y = , and standard error of slope =

13. A Methanolysis samples (s) of tannic acid
B . EGCG
C. Acer leaf powder
Conversion of hydrolyzable tannins to methyl gallate by 20 h of methanolysis at 85°C.

14. Theoretical Production of Methyl Gallate from Hydrolyzable Tannins
Yield of methyl gallate is calculated from molecular weights of tannin and
methyl gallate and is expressed in grams of methyl gallate per gram of tannin.

15. Example:red maple leaves
Analysis was done with the original KIO3 method (A)
Analysis was done with the new method described (B)

16. Theoretical Production of Methyl Gallate from Hydrolyzable Tannins
Yield of methyl gallate is calculated from molecular weights of tannin and
methyl gallate and is expressed in grams of methyl gallate per gram of tannin.

17. Conclusion
The new KIO3 method will be useful for examining hydrolyzable tannin in ecological, physiological, and nutritional studies of plants.