ANALYSIS OF HERBAL

DRUGS BY TITRIMETRY

Prepared by-

Vedshree Raole

Guided by-

Dr.S.J Rajput

Faculty of Pharmacy,

M. S. University

CONTENTS

Definitions

Introduction to Titrimetry

Estimation of Marketed Extracts.

Estimation of Marketed tablets.

Estimation of Excipients in herbal

formulation

Estimation of Other Marketed

Formulations.

References

Definitions

Crude herbs: This term means, unless specified

otherwise, mainly whole, fragmented or cut, plants,

parts of plants, algae, fungi, and lichen in a form which

is not processed.

Processed herb: Processed herbs means preparations

obtained by subjecting herbs to treatment such as

extraction, distillation, expression, fractionation,

purification, concentration and partial or full

fermentation.

Dry Extract: Dry extracts usually have a loss on drying

or water content not greater than 5 per cent w/w,

unless specified otherwise in any monograph.

Tincture: would normally mean an extract where

aqueous-ethanol is used as a solvent for extraction.

Standardization as per WHO: Measures

taken during the manufacturing and

Quality control leading to the

reproducible quality.

It can be achieved by adjusting the extract

with approved inert materials or blending

one or more batches of extracts.

TITRIMETRY

Titration is a method for determining the concentration of a solution by

reacting a known volume of that solution with a solution of known

concentration.

A neutralization reaction is a reaction in which an equal molar quantities of

acid and a base in an aqueous solution react to produce a salt and water.

Complexometric titration (chelatometry) is a form of volumetric analysis in

which the formation of a colored complex is used to indicate the end point of

a titration.

Non- aqueous titrations are the titrations in which weakly acidic or basic

substances are carried out using non–aqueous solvents to get sharp end

point.The moisture content in non–aqueous titrations should not be more

than 0.05%.

Redox reactions are the oxidation-reduction reactions in which a

change in the valency of reacting elements takes place.Titrations in

which titrant and analyte undergo redox reactions.

Estimation of Tannins in

Amla Juice powder[1]

Weigh accurately about 2 g of the extract, add 50 ml boiling water and heat

it on a water-bath for 30 minutes with frequent stirring. Allow the solution

to settle and carefully decant it through a piece of cotton wool to a 500-ml

volumetric flask.

Repeat the extraction for 5 times with 50 ml of boiling water. To confirm

that all tannins have been extracted, add 3-4 drops of ferric ammonium

sulphate solution to 5 ml of the extract. Blue colour will not be produced, if

all tannins have been extracted. If blue colour develops extract again with 2

times with 50 ml of boiling water and check with ferric ammonium sulphate

solution again. Cool the extracts and make up to the mark with water.

Take 50 ml into a 250-ml conical flask and add 50 ml of indigo sulphonic

acid solution(prepared by dissolving 1 g of indigo carmin in 50 ml of

sulphuric acid, add 500 ml of water and dilute this solution to 1000 ml.)

Titrate, with 0.1 M KMnO4 using indigo sulphonic acid as indicator until a

golden yellow colour is produced. Carry out a blank titration.

1 ml of 0.1 M KMnO4 is equivalent to 0.004157 g of polyphenoIs calculated

as tannic acid.

Estimation of dried

Belladona Leaf tincture[1]

Evaporate 50.0 g of the tincture under examination to a volume of

about 10 ml. Transfer quantitatively to a separating funnel, with the

minimum volume of ethanol (70 per cent v/v). Add 5 ml of strong

ammonia solution and 15 ml of water.

Extract with three quantities, each of 40 ml of a mixture of 1 volume of

dichloromethane and 3 volumes of peroxide-free ether, carefully to

avoid emulsion, if the alkaloids are completely extracted. Combine the

dichloromethane and ether extracts; concentrate the solution to a

volume of about 50 ml by heating on a water-bath. Transfer the

resulting solution quantitatively to a separating funnel, rinsing with

peroxide-free ether.

Add a quantity of peroxide-free ether equal to at least 2.1 times the

volume of the solution to produce a layer having a density well below

that of water. Extract the resulting solution with minimum of three

quantities, each of 20 ml of 0.25 M sulphuric acid until the alkaloids are

completely extracted.

Separate the layers by centrifugation if necessary and transfer the layers

to a separating funnel. Make the combined layers alkaline with strong

ammonia solution and extract with minimum of three quantities, each of

30 ml of dichloromethane until the alkaloids are completely extracted.

Combine the dichloromethane and ether extracts, add 4 g

of anhydrous sodium sulphate and allow standing for 30

minutes with occasional shaking. Decant the

dichloromethane and filter. Wash the sodium sulphate

with three quantities, each of 10 ml of dichloromethane.

Combine the dichloromethane and ether extracts;

evaporate to dryness on a water-bath. Heat the residue

in an oven at 105° for 15 minutes.

Dissolve the residue in a few ml of dichloromethane,

evaporate to dryness on a water-bath and heat the

residue in an oven at 105° for 15 minutes again. Dissolve

the residue in a few ml of dichloromethane. Add 20.0 ml

of 0.01 M sulphuric acid and remove the

dichloromethane by evaporation on a waterbath.

Titrate the excess of acid with 0.02 M sodium hydroxide

using methyl red mixed solution as indicator.

1 ml of 0.01 M sulphuric acid is equivalent to 0.005788 g

of total alkaloids calculated as hyoscyamine.

Calculate the content of total alkaloids with reference to

the dried material.

Estimation of Calcium in

Garcinia Aq. Extract[1]

Calcium -

Test solution. Weigh accurately about 0.2 g of

the extract and transfer into a 500 ml conical

flask. Dissolve in 2 ml of 3 M hydrochloric

acid and add 200 ml of water. Add 15 ml of 1

M Sodium hydroxide and titrate with 0.05 M

EDTA using hydroxy naphthol blue as

indicator until deep blue colour persist.

Each ml of 0.05MEDTA solution is equivalent

to 0.002004 g of calcium.

Estimation of Papain in

Carica Papaya extract[1]

Weigh accurately about 0.5 g, triturate with 10 ml of

cysteine hydrochloride solution and dilute to 100.0 ml

with water.

To 30 ml of water in each of two flasks add 15.0 ml of

casein solution and maintain at 60° by heating on a

water-bath.

To the first flask add 5 ml of solution under

examination and to the second flask- add 5.0 ml of

the same solution, previously boiled for 2 minutes and

cooled. Maintain the solutions at 60° for 30 minutes,

cool rapidly to room temperature.

Add to each flask 0.75 ml of phenolphthalein solution

and 10 ml of formaldehyde solution, previously

neutralised to phenolphthalein solution.

Titrate both solutions with 0.1 M sodium hydroxide

to the same definite pink colour; the difference

between the two titrations is not less than 4.5 ml.

Estimation of Peppermint

Oil[1]

For esters –

To 2 g in a borosilicate glass flask add 2 ml of ethanol (90

per cent) and 0.25 ml of phenolphthalein solution,

neutralise with 0.5M ethanolic potassium hydroxide.

Add an additional 25.0 ml and a little pumice powder or

a few pieces of porous pot and heat under a reflux

condenser on a water-bath for 30 minutes.

Add 1 ml of phenolphthalein solution and immediately

titrate with 0.5M hydrochloric acid.

Repeat the operation without the substance under

examination. The difference between the titrations

represents the volume of alkali required to saponify the

esters.

1 ml of 0.5 M ethanolic potassium hydroxide is equivalent

to 0.09915 g of esters, calculated as menthyl acetate,

CI2H

220

2.

For free alcohols –

To 1 g in a dry, 150-ml acetylation add 3 ml of a mixture of 3

volumes of pyridine and 1 volume of acetic anhydride. Determine

the weight of the acetylation mixture to the nearest mg, keeping the

flask closed while weighing.

Boil under a reflux condenser in a water-bath for 3 hours,

maintaining the water level 2 to 3 cm above the level of the liquid in

the flask throughout. Remove the flask from the water-bath and add

50 ml of water through the condenser. Remove the condenser and

wash the walls of the flask with 10 ml of water.

Allow to stand for 15 minutes and titrate with 0.5 M sodium

hydroxide using 1 ml of phenolphthalein solution as indicator. Repeat

the operation without the substance under examination.

The difference between the titrations represents the volume of

sodium hydroxide required.

1 ml of 0.5 M sodium hydroxide is equivalent to 0.07815 g of free

alcohols, calculated as menthol, CIOH2oO.

If the quantities of acetic anhydride in pyridine used in the two

determinations differ by more than 5 mg, adjust the volume of alkali

used in the second titration by multiplying with a/b where a is the

weight, in g, of acetic anhydride in pyridine used in the first

determination and b is the weight, in g, of acetic anhydride in

pyridine used in the second test.

For ketones-

•To 2 g add 25 ml of a 5.0 %w/v solution of hydroxylamine

hydrochloride in ethanol (95 per cent), heat on a water-bath for 1 hour,

allow to cool, add about 1mg of methyl orange.

•Titrate with 0.5 M ethanolic KOH until an orange-yellow colour is

obtained.

•Repeat the heating for further periods of 1 hour until, after cooling, not

more than 0.1 ml of 0.5 M ethanolic potassium hydroxide is required to

neutralise the solution.

•1 ml of 0.5 M ethanolic potassium hydroxide is equivalent to 0.07710 g

of ketones, calculated as menthone, CIOH1SO.

Estimation of Acid value

in Shellac[1]

Acid value (2.3.23). 50 to 70, determined by the following method.

• Weigh accurately about 2.0 g and dissolve with the aid of gentle heat, in 50 ml

of ethanol (95per cent) previously neutralised to ethanolic thymol blue solution.

•Titrate with 0.1 M ethanolic potassium hydroxide using ethanolic thymol blue

solution as an external indicator.

• Calculate the acid value from the expression

5.61 x a/w

where, a number of ml of 0.1 M ethanolic potassium

hydroxide and

w = weight, in g, of the sample.

Estimation of Ephedrine in

marketed formulation[2]

Weigh 20 tablets and reduce to a fine powder.

Weigh accurately a quantity of the powder

equivalent to about 0.15 g of Ephedrine

Hydrochloride and add 30 ml of anhydrous

GAA and 10 ml of mercuric acetate solution.

Warm gently to effect solution, cool and for

non-aqueous titration, using 0.1 ml of crystal

violet solution as indicator, until the violet

colour changes to green-blue.

Perform a blank determination and make any

necessary correction.

Each ml of 0.1M perchloric acid is equivalent to

0.02017 g of C10

H15

NO,HCl.

Estimation of cinnamic acid

and benzoic acid in tolu

balsam marketed preperation[2]

Weigh accurately about 1.25 g and boil with 25 ml of dilute

ethanolic potassium hydroxide solution under a reflux condenser for

1 hour. Remove the ethanol and digest the residue with 50 ml of hot

water until diffused. Cool the liquid, add 150 ml of water and 1.5 g

of magnesium sulphate dissolved in 50 ml of water.

Mix thoroughly and set aside for 10 minutes. Filter, wash the residue

on the filter with 20 ml of water, acidify the combined filtrate and

washings with HCl and extract with successive quantities of 50, 40,

30, 30 and 30 ml of ether.

Combine the ether extracts and discard the aqueous portion. Extract

with successive quantities of 20, 20, 10, 10 and 10 ml of sodium

bicarbonate solution, washing each aqueous extract with the same

20 ml of ether. Discard the ether layers, acidify the combined

aqueous extracts with hydrochloric acid and extract with successive

quantities of 30, 20, 20 and 10 ml of chloroform, filtering each

chloroform extract through a plug of cotton wool on which a layer

of anhydrous sodium sulphate is placed.

Evaporate the chloroform on a water-bath until

about 10 ml remains and remove the remainder in a

current of air stopping immediately when the last

trace of solvent is removed.

Dissolve the residue by warming with 10 ml of

ethanol (95%), previously neutralised to phenol red

solution, cool and titrate with 0.1M sodium

hydroxide using phenol red solution as indicator.

Each ml of 0.1M sodium hydroxide is equivalent to

0.01482 g of total balsamic acids,calculated as

cinnamic acid, C9H

8O

2, in Sumatra Benzoin and

0.01221 g of total balsamic acids, calculated as

benzoic acid, C7H

6O

2, in Siam Benzoin.

Estimation Alkaloid

content in Kutuja[2,4]

Kutaja contains NLT 2% of total alkaloids when assayed by the

following method:

Weigh accurately about 5 g in powder (No. 85 sieve) and moisten with

10 ml of an Alcohol-chloroform mixture (1 :3) containing 2% of

Ammonia solution for 15 minutes.

Pack the mixture in a small glass percolator surrounded by a jacket of

hot water kept at 50°. Macerate with more of the alkaline Alcohol-

chloroform mixture for an hour and collect 25 ml of percolate in a

receiver containing 1 g of Oxalic acid dissolved in 5 ml of alcohol.

Stop the percolation add l0 ml of the alcohol chloroform mixture

containing 1% w/v of NaOH and macerate for fifteen minutes. Continue

the percolation adding further quantities of the alcohol chloroform

mixture until the alkaloids are completely extracted. Mix the percolate

well and extract by shaking with five 20 ml portions of 2 N

Hydrochloric acid. Combine the acid extracts and make alkaline with

dilute Ammonia Solution.

Extract with four 10 ml portions of Chloroform, add

1 ml of 0.5 N Sodium Hydroxide, and extract again

with Chloroform. Wash each Chloroform extract

with the same two 10 ml portions of water

contained in different separators.

Combine the Chloroform extracts, add 20 ml of

O.IN Sulphuric Acid and shake well for 5 Minutes.

Transfer the acid Liquid to a conical flask, wash the

Chloroform extract with two 20 ml portions of

water and add the washing to the acid liquid in the

conical flask.

Titrate the excess of acid with 0.1N Sodium

Hydroxide using the mixed 3 indicator.

Each ml of 0.1N Sulphuric Acid is equivalent to

0.01657g of total alkaloids of Kutaja.

Estimation of Strychnin

in Strychnous Nux

Vomica[2]

Weigh accurately about 10g in fine powder, add 100 ml of

a 33% v/v mixture of chloroform in solvent ether and set

aside for ten minutes.

Add 5 ml of dilute ammonia solution and shake

continuously for six hours. Transfer to a continuous

extraction apparatus with more of the same solvent

mixture and extract for two hours.

Filter the solvent extract, washing the filter with solvent

ether and extract with successive quantities of 20 ml, 20

ml,10 ml and 10 ml of 1N sulphuric acid, until complete

extraction of the alkaloids is affected. Combine the acid

extracts and make alkaline with dilute ammonia solution.

Extract with successive quantities of 20 ml, 20 ml and 10 ml

of chloroform until complete extraction of the alkaloids is

effected. Evaporate the chloroform, add 5 ml of alcohol

and evaporate to dryness.

Dissolve the residue in a mixture of 15 ml of a 3% w/v solution of

sulphuric acid and 2 ml of nitric acid, add a few crystals of sodium nitrite

and set aside at 18°C for thirty minutes.

Transfer to a separator containing 20 ml of solution of NaOH, shake for

two minutes and then shake with 20 ml of chloroform, separate the

chloroform solution, wash it with 5 ml of solution of NaOH and then

with two quantities each of 10 ml of water.

Continue the extraction with successive quantities of 10 ml of

chloroform, until complete extraction of the alkaloids is effected,

washing each chloroform solution separately with the 5 ml of solution of

sodium hydroxide and with the two quantities of water, which were

used for washing the first chloroform solution.

Titrate the second wash with 0.1 N sulphuric acid using solution of

methyl orange as indicator if more than 0.1 ml is required, wash the

combined chloroform solutions with further quantities, each of 10 ml of

water until on titration not more than 0.1 ml of 0.1 N sulphuric acid is

required.

Remove the chloroform, add 5 ml of alcohol, evaporate, and dry for

thirty minutes, at 100°C. Dissolve the residue in 10 ml of 0.1 N sulphuric

acid and titrate the excess of acid with 0.1 N sodium hydroxide, using

solution of methyl orange as indicator.

Each ml of 0.1 N sulphuric acid is equivalent to 0.03344 g of strychinine,

multiply the result by 1.02 to correct for loss of strychinine.

Presence of sodium benzoate in

commercially available samples

of Dasamoolarishta[13]

Benzoic acid was separated from a known quantity of sample

by saturating with NaCl, and acidifying with dilute HCl.

This sample was extracted with chloroform. The chloroform

layer was made mineral acid free and the solvent was

removed by evaporation.

The residue was dissolved in neutral alcohol and the amount

of benzoic acid determined by titration against standard alkali.

The amount of benzoic acid present in the sample was

determined using the formula

Benzoic acid (ppm) = 122 X titre X dilution X 1000 X NaoH

Weight of sample

STANDARDIZATION OF

PANCHATIKVA GHRITA [8]

Acid Value:

10gm of sample is taken in a 250 ml volumetric flask and 50ml of

mixture of equal volumes of ethanol and solvent ether are added and

is neutralised after addition of 1 ml of phenolphthalein solution.

This is heated gently on water bath until the substance is completely

melted.

It is then titrated against 0.1N potassium hydroxide solution with

continuous shaking until pink color which is persistent for 15 seconds

is obtained.

Number of ml is noted and calculation is done using the following

equation:

Acid value= a×0.00561 ×1000/W

where,

a= number of ml of 0.1N KOH required

W= Weight of sample in gm

Iodine Value:

Wij’s method is used for determination of Iodine value.

10ml of iodine monochloride is mixed with 1800 ml of GAA and shaken vigorously.

2g of sample is taken in Erlenmayer flask, 25 ml of carbon tetrachloride is added and

the content is mixed well. To this 25ml of Wij’s solution is pipetted and the flask is

stoppered with glass stopper previously washed with KI solution.

It is swirled for proper mixing and kept in dark for 1 hour. Blank is prepared in the

same manner.

15ml of KI is added followed by 100 ml of freshly boiled and cooled water. Rinsing of

stopper is also done.

The liberated iodine is titrated against standardised sodium thiosulphate solution using

starch indicator until persistent blue color is obtained.

Iodine value is calculated as:

Iodine value= 12.69(B-S) N/W

where,

B = ml of sodium thiosulphate required by blank

S = ml of sodium thiosulphate required by sample

N = Normality of sodium thiosulphate

W = Weight of sample taken

Quantitative Estimation of

tannin (gallotannic acid) in

marketed ayurvedic churna

preparations [5]

Pipette out 10ml from the stock solution and add 10ml indigo carmine

indicator solution and make up volume up to 100ml with distilled

water. Heat this solution on water bath for 15-20 min at 70oc and

titrated against 0.1N KMnO4 solution which has taken in burette. Note

the Colour changes from blue to parrot green and finally the

appearance of golden yellow color at end point. The consumed

volume of 0.1N KMnO4 is recorded as A. Take another 2 reading using

same procedure and take mean out of that. Blank reading taken by

replacing sample solution with distilled water and follow the same

procedure as above. Note down the ml of KMnO4 is used in blank as

B

%Tannins = (A - B)*250 *100*(0.00425 gm tannins/ml of 0.1N KMnO4)

gm of sample powder * ml of sample taken

Determination of ethanol

content of the Abhayarishta by

Redox titration [7]

The method uses a redox titration to find the concentration of

ethanol in the aqueous solution.

The ethanol gets oxidized to ethanoic acid by reacting it with excess

of potassium dichromate in concentrated sulphuric acid.

Transfer 10 mL of the acid dichromate solution to a 250 mL conical

flask with matching rubber stopper.

Pipette 1 mL of the diluted sample into the sample holder. This can

be a 5mL beaker or glass vial. Prepare three samples of the beverage

as the entire contents of the flask are used in the titration.

Suspend the sample holder over the dichromate solution and hold in

place with the rubber stopper.

Store the flask overnight at 25–30°C (an incubator is ideal).

Next morning allow the flask to come to room temperature, then

loosen the stopper carefully and remove and discard the sample

holder.

Rinse the walls of the flask with distilled water,

then add about 100 mL of distilled water and 1 mL

of potassium iodide solution. Swirl to mix.

Prepare 3 blank titrations by adding 10 mL of acid

dichromate solution to a conical flask, adding 100

mL of water and 1 mL of potassium iodide solution

and swirling to mix.

Fill a burette with sodium thiosulfate solution and

titrate each flask with sodium thiosulfate. When

the brown iodine colour fades to yellow , add 1

mL of starch solution and keep titrating until the

blue colour disappears Titrate the blank flasks first,

and repeat until concordant results.

Determination of reducing

sugars in

Marketed Kumari Asava[11]

20ml of kumari asava was taken and neutralize with

NaOH. The neutralize solution was evaporated to

half volume on waterbath at 50˚C to removed

alcohol. After cooling 10 ml of 21.9 g zinc acetate,

3ml GAA followed by 10.6 g potassium ferrocyanide

and distilled water was added to make a volume of

100ml.

10ml of Fehling solution was taken and burette

solution was added drop wise and heat to boiling

over hot plate till blue color appeared. At this time,

two drops of methylene blue was added and the

titration was carried on till brick red color was

obtained.

References

1. The Indian pharmacopoeia, IP ‘10, volume-3, Indian

pharmacopoeia commission, Ghaziabad, India, 1996. Pg No.:

744-830

2. Ayyurvedic pharmacopoeia Vol-I part-1 Pg no:119-120, part-4 Pg

No:170-171.

3. WHO guidelines for assessing quality of herbal medicines with

reference to contaminants and residues

4. Identification, evaluation and standardization of herbal drugs: A

review Archana Gautam*, Shiv Jee

Kashyap(http://scholarsresearchlibrary.com/archive.html)

5. Validated simple redox titration method for the estimation of

gallotannins in marketed ayurvedic churna preparations Ankit V.

Patel*, Kalpen N. Patel and Maulika S. Patel J. Chem. Pharm.

Res., 2011, 3(6):293-299

6. Development of Evaluation Parameters for Balchaturbhadra Churna &

Comparision with Market Formulation Shahebaz N. Ghadiyali*, Nitesh

Patel, Nikunj Trivedi , Hetal Desai, IJPRAS Volume 1, issue 4 (2012),75-84

7. STANDARDIZATION OF ABHYARISHTA AS PER WHO GUIDELINES

Shreyansh S. Mutha, Veena S. Kasture*, Seema A. Gosavi, Rasika D

Bhalke.,Sarita S. Pawar WJPPS Volume 3, Issue 1, 510-518.

8. Pharmacoanalytical study and Standardization of Panchatikta Gritha,

Haldar Pranob, IRJP 2013,4(9)

9. http://www.outreach.canterbury.ac.nz/chemistry/ethanol.shtml

10. Pharmaceutical Drug Analysis by Ashutosh Kar

11. STANDARDIZATION OF MARKETED KUMARI ASAVA- A POLYHERBAL

AYURVEDIC FORMULATION DS. Chumbhale1*, SR. Chaudhari1 and CD.

Upasani IJPCBS 2014, 4(3), 681-685

12. DETERMINATION OF TANNINS CONTENT BY TITRIMETRIC METHOD

FOR COMPARISON OF DIFFERENT PLANT SPECIES M. Atanassova, V.

Christova-Bagdassarian

13. A study on the presence of sodium benzoate in commercially available

samples of Dasamoolarishta- an ayurvedic preparation Nishna KP, Robin

PC, Harikumar R and Jayachandran VP*, IJPCS, ISSN: 2277-5005.

Estimation of Acidity in Starch Add 10.0 g to 100 ml of ethanol (70 per cent)

previously neutralised to phenolphthalein

solution, shake for 1 hour, filter.

Titrate 50 ml of the filtrate with 0.1 M sodium

hydroxide.

Not more than 2.0 ml is required to change the

colour of the solution.