Chemical tests, isolation and estimation of Tannins

Content

Chemical tests, isolation and estimation of Tannins

Objective

At the end of this lecture, student will be able to

• Identify tannins by chemical tests

• Describe the different methods of isolation and estimation of tannins

Identification tests for Tannins

• Match stick test

• Gelatin test

• Phenazone test

• Chlorgenic acid test

• Gold beaters skin test

Match stick test (Catechin test)

Dip a match stick in aqueous plant extract, dry near burner and moisten with Hcl. On warming near flame, the match stick wood turns magenta or purple due to the formation of phloroglucinol

Gelatin test

Solution of tannin (0.5%-1%) precipitates 1% solution of gelatin containing 10% sodium chloride

Phenazone test

To 5ml of aqueous extract of drug, add 0.5grams of sodium acid phosphate, warm it and cool. Filter the solution and to the filtrate add 2% solution of Phenazone. Tannins will be precipitated as bulky ppt

Test for chlorogenic acid

Treat extract containing chlorogenic acid with aqueous ammonia and expose it to air. Green color will appear gradually

Gold beater’s skin test

Gold beater’s skin is a membrane prepared from intestine of Ox

Soak a small piece of Gold beater’s skin in 2% hydrochloric acid. Rinse it with distilled water. Place it in a solution to be tested for 5 minutes. Wash in water and transfer to 1% solution of ferrous sulphate. Black or brown color of skin indicates presence of tannins

It is a quantitative test and +ve only for true tannins

Isolation of Tannins

• Both hydrolysable and condensed tannins are highly soluble in water and alcohol but insoluble in organic solvents such as chloroform, and benzene

• Hence, tannin compounds can be easily extracted by water or alcohol

• The general method for the extraction of tannins is either with water-saturated ether, or with mixture of water, alcohol, and ether

• In such cases, free acids such as gallic and ellagic acid go along with ether while true tannin gets extracted in water

• If the drug consists of chlorophyll or pigment, it may be removed by ether

• After extraction, the aqueous and ethereal layers are separately concentrated, dried

• Further isolation and purification is carried out using various separation techniques of chromatography

Estimation of Tannins

Tannins are estimated as

• Total tannins

• Tannic acid equivalent

• Ellagitannin content

• Proanthocyanidin content

• Gallotannin content

Total tannins:

• Carefully dried drug is finely powdered

• Weighed amount is extracted with 3 portions successively with aqueous methanol (1:1) using about 0.1 ml solvent / mg

• One or more of the following procedures may be used depending on what type of tannins are present

- Tannic acid equivalent

- Proanthocyanidin content

- Ellagitannin content

- Gallotannin content

Tannic acid equivalent:

1 ml extract + 1 ml fresh sample of diluted human blood (1:50)

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Centrifugation

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Tannin protein ppt

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Residual hemoglobin (578 nm)

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Standard measurements of tannic acid

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Astringency of plant extract

Proanthocyanidin content:

A known volume of extract is concentrated to 1/3 volume

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0.5 ml of extract + 4 ml of n-butanol containing 5% con HCl

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Heat at 95 ⁰C for 2 hrs

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Absorbance at 545 nm (cyanidin) and 560 nm (delphinidin)

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Comparison with Std proanthocyanidin

Ellagi tannin content:

0.5 ml of extract + 2 ml of 0.1N HNO2 (NaNO2 and acetic acid)

â Room temp

Blue colour (600 nm, after 15 min)

Gallitannin content:

0.5 ml of extract + 1.5 ml of 12 % KIO3 in 33% CH3OH, 15 ⁰C

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Red brown colour (550 nm)

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Galloyl groups

Summary

Identification –

- Match stick test (Catechin test),

- Gelatin test

- Phenazone test

- Chlorgenic acid test

- Gold beater skin test

• Extraction with water or alcohol

Estimation

- Tannic acid equivalent

- Ellagitannin content

- Proanthocyanidin content

- Gallotannin content