Tannic acid is widely used in fields such as food, pharmaceuticals, leather production, and animal husbandry, and the detection technologies associated with it are increasingly attracting attention. Currently, the main methods for determining tannic acid include spectrophotometry, titration, protein pre-precipitation, hide powder method, high-performance liquid chromatography, and fluorescence analysis. Below is an analysis of the advantages and disadvantages of each detection method:

Spectroscopy

• NYSL-1006-2022 Determination of Tannic Acid in Feed by Spectrophotometry (Currently in effect)
• LY/T 1642-2005 Tannic Acid Analysis Method: UV Spectrophotometry (Currently Valid)
• GB/T 27985-2011 Determination of Tannins in Feed by Spectrophotometry (Currently Valid)
• GB/T 15686-2008 Determination of Tannin Content in Sorghum (Currently Valid)

1. LY/T 1642-2005 The method works by using a UV spectrophotometer to measure the absorbance of both the sample solution and the non-tannin solution (after tannins have been removed using chrome leather powder) at a wavelength of 276 nm. The difference in absorbance values, when compared to the absorbance of a standard tannic acid solution, allows for the calculation of the tannin content in the sample—though this result is significantly influenced by the choice of standard reference material.

2. NYSL-1006-2022 and GB/T 27985-2011 The method for determining tannic acid primarily involves: extracting tannin compounds from the feed using an acetone solution, followed by filtration. The resulting filtrate is then mixed with a sodium tungstate-phosphomolybdic acid solution and sodium carbonate solution. After developing the color (due to an oxidation-reduction reaction), the absorbance is measured at 760 nm using a spectrophotometer, with water serving as the blank control. Finally, the tannin content is quantified by constructing a standard curve using tannic acid reference material (galla chinensis).

3. NYSL-1006-2022 It includes one additional step compared to GB/T 27985-2011—specifically, hydrochloric acid must be used to break the chemical bonds of tannin-protein complexes, releasing the tannins before proceeding with the analysis.

4. GB/T 15686-2008 It belongs to the precipitation method based on protein-binding properties and can be seen as a variation or application of the vanillin method.

Advantages: All four methods work. Fast, convenient, and widely applicable—results are typically available within a single day.

Drawbacks 1. Method NYSL-1006-2022 And methods GB/T 27985-2011 The measurement results may be influenced by reducing non-phenolic substances (such as vitamin C, reducing sugars, and sulfites), potentially leading to inaccuracies in determining only the tannin content. NYSL-1006-2022、 LY/T 1642-2005 and GB/T 27985-2011 Currently, only a pentaphylla standard is available on the market among the reference standards mentioned. Other sources of tannic acid standards are not yet commercially accessible, so laboratories must prepare their own internal reference standards for comparison. As a result, the accuracy of test results can be significantly influenced by both the type of standard used and the precision of the tannic acid content measurement.

          2. Method GB/T 15686-2008 Color development is unstable: The resulting red substance is highly unstable and must be measured within a specified time—50 minutes according to the standard—making precise timing critical for accurate results.

Titration method

Potassium Permanganate Titration Method: As a type of polyphenol, tannins are strong reducing agents that are highly susceptible to oxidation by oxidizing agents and can also be adsorbed by activated carbon. In this method, potassium permanganate is used as the oxidizing agent. By measuring the difference in oxidation values before and after the sample solution is treated with activated carbon—since tannins in the sample are adsorbed by the carbon—the content of tannin substances can be quantified.

Advantages: One of the classic methods, relatively simple to perform, is suitable for the complexation reaction between various metal ions and tannic acid.

Drawbacks: Precise control of titration conditions is required, and the method may lack sensitivity for certain samples with low tannin content.

Protein Precipitation Method

Protein Precipitation Method: Tannic acid possesses a polyphenol structure and can bind to proteins, forming a precipitate. Tong Jian [7] et al. quantitatively precipitated tannins using bovine serum albumin, then dissolved the resulting precipitate in an aqueous solution of sodium dodecyl sulfate-triethanolamine (SDS-TEA). Upon reaction with a sodium chloride reagent, a red complex was formed, whose absorbance value exhibited a linear relationship with the tannic acid concentration in the range of 0–50 μg/mL. Experimental results showed that the recovery rate of tannic acid ranged from 98.16% to 100.66%.

Advantages: By leveraging the property of tannic acid to form a precipitate when it binds with proteins, the method allows for the simple, rapid, and accurate determination of tannic acid content through absorbance measurements.

Drawbacks: Specific proteins and conditions may be required to ensure complete and reproducible precipitation.

Gravimetric Analysis Method – Peel Powder Technique

The pyrophoric method is an internationally recognized technique for tannin analysis. The mass fraction of dry matter in the filtrate, after tannins have been removed from the test solution using chrome leather powder, represents the non-tannin content. The difference between the soluble solids content and the non-tannin content equals the tannin level.

LY/T 1082-2021 Experimental Methods for Tack Gum Raw Materials and Products

GB/T 38410-2019 — Leather Chemicals: Determination of Tannin Content in Tanning Agents — Filtration Method

Advantages: The Pifan method has a wide range of applicability and demonstrates good reproducibility under rigorous operational conditions. This method accurately distinguishes between the contents of tannin and non-tannin components.

Drawbacks: The sample consumption is high, the measurement takes a long time, and it’s impossible to distinguish between the individual contents of hydrolyzable tannins, gallic acid tannins (ellagitannins), and condensed tannins.

High-Performance Liquid Chromatography

Guo Jiali and colleagues used high-performance liquid chromatography to determine the tannic acid content, employing a Hypersil ODS column (125 × 4.0 mm, 5 μm) with a mobile phase consisting of 80% CH3OH and 20% H2O. The analysis was conducted at a detection wavelength of 275 nm and a flow rate of 0.5 mL/min. The method exhibited a linear range of 120–240 ng/mL. When applied to measure tannic acid in samples of Terminalia chebula extract, the spiked recovery rates ranged from 99.7% to 107.5%. Moreover, each single analysis could be completed within just 3 minutes, making this method highly efficient for quantifying tannic acid in a variety of substances. Ke Weichang and colleagues used automated solid-phase extraction to remove interfering components, followed by reversed-phase high-performance liquid chromatography to determine tannic acid in concentrated extracts from paper-based reconstituted tobacco leaves. This method is simple, rapid, and highly accurate. It exhibits a good linear relationship over the tannic acid concentration range of 2–117 μg/mL, with a detection limit of 0.2 μg/mL.

Zhang Xueling and colleagues selected the Ultimate AQ-C18 column (250 mm x 4.6 mm, 5 μm) and conducted a comparative analysis using three mobile phases: methanol-0.1% formic acid water, acetonitrile-0.1% formic acid water, and acetonitrile-0.1% phosphoric acid water. The results demonstrated that when the Ultimate AQ-C18 column was used with a gradient elution system employing acetonitrile and 0.1% phosphoric acid solution as the mobile phase, effective separation from interfering compounds could be achieved. Under these conditions, the tannic acid standard exhibited a single, symmetric peak with a relative retention time of approximately 11.910 minutes, showing excellent reproducibility.

South Kun Bio Company Select the Zhongpu Red RD-C18 (250 mm x 4.6 mm, 5 µm) chromatographic column, and use a methanol + water (D) mobile phase for gradient elution with the following program: 0.01 D.Conc  90%; 5.00 D. Conc   70%; 6.00 D.Conc 40%; 20.00 D.Conc 0%; 22.00 Stop, The results show that using a Zhongpu Red RD-C18 chromatographic column with methanol and water as the mobile phase in gradient elution effectively achieves separation from interfering components. Under these conditions, the tannic acid standard exhibited a single, symmetric peak with a relative retention time of approximately 12.127 minutes, demonstrating excellent reproducibility.

Advantages: It can provide precise quantification of tannic acid with high resolution, enabling the simultaneous determination of multiple components.

Drawbacks: The equipment is relatively expensive and requires professional operation and maintenance.

Fluorescence Analysis Method

Feng Suling and colleagues have developed a fluorescence kinetic method for the determination of tannic acid, which relies on the activation of rhodamine B oxidation by hydrogen peroxide catalyzed by Cu(II) in the presence of tannic acid. The method exhibits a linear range of 0.04–0.72 mg/L and a detection limit as low as 0.025 mg/L. Through systematic investigation of various reagent concentrations, the optimal experimental conditions were carefully selected. When applied to a tannic acid standard solution with a concentration of 0.4 mg/L, the method yielded 11 parallel measurements with an RSD of 1.8%. Furthermore, when analyzing processed tea samples, the recovery rates ranged from 95.2% to 104%, with an RSD of 1.7% to 3.0%.

Advantages : Highly sensitive, capable of detecting extremely low concentrations of tannic acid.

Drawbacks: It may require specific fluorescent labels and complex sample preparation.