Limit Test

Limit test is defined as quantitative or semi quantitative test designed to identify and control smallquantities of impurity which is likely to be present in the substance.

Limit test is generally carried out to determine the inorganic impurities present in compound.

Limit test is nothing butto identify the impurities present in the substance and compare it with standard.

Limit = a value or amount that is likely to be present in asubstance

Test = to examine or to investigate

Impurities = a foreign matter present in a compound

Importance of Limit tests

To find out the harmful amount of impurities

To find out the avoidable/unavoidable amount of impurities.

Limit test for Chlorides

Principle:

Limit test of chloride is based on the reaction of solublechloride with silver nitrate in presence of dilute nitric acid to form silver chloride, which appears as solid particles (Opalescence) in thesolution.

Procedure:

Test sample	Standard compound
Specific weight of compound is dissolved in water or solution is prepared as directed in the pharmacopoeia and transferred in Nessler cylinder	Take 1ml of 0.05845 % W/V solution of sodium chloride in Nessler cylinder
Add 1ml of nitric acid	Add 1ml of nitric acid
Dilute to 50ml in Nessler cylinder	Dilute to 50ml in Nessler cylinder
Add 1ml of AgNO3 solution	Add 1ml of AgNO3 solution
Keep aside for 5 min	Keep aside for 5 min
Observe the Opalescence/Turbidity	Observe the Opalescence/Turbidity

Observation:

The opalescence produce in sample solution should not be greater than standard solution.

If opalescence produces in sample solution is less than the standard solution, the sample will pass thelimit test of chloride and visaversa.

Reasons:

Nitric acid is added in the limit test of chloride to make solution acidic and helps silver chloride precipitate to make solution turbid at the end of process.

Limit test for Sulphates

Principle:

Limit test of sulphate is based on the reaction of soluble sulphate with barium chloride in presence of dilute hydrochloric acid to form barium sulphate which appears as solid particles (turbidity) in thesolution.

Procedure:

Test sample	Standard compound
Specific weight of compound is dissolved in water or solution is prepared as directed in the pharmacopoeia and transferred in Nessler cylinder	Take 1ml of 0.1089 % W/V solution of potassium sulphate in Nessler cylinder
Add 2ml of dilute hydrochloric acid	Add 2ml of dilute hydrochloric acid
Dilute to 45 ml in Nessler cylinder	Dilute to 45 ml in Nessler cylinder
Add 5ml of barium sulphate reagent	Add 5ml of barium sulphate reagent
Keep aside for 5 min	Keep aside for 5 min
Observe the Turbidity	Observe the Turbidity

Bariumsulphate reagent contains barium chloride, sulphate free alcohol and small amount of potassium sulphate.

Observation:

Theturbidity produce in sample solution should not be greater than standard solution. If turbidity produces in sample solution is less than the standard solution, the sample will pass the limit test of sulphate and vice versa.

Reasons:

Hydrochloric acid helps to make solution acidic. Potassium sulphate is used to increase the sensitivity of thetest by giving ionic concentration in the reagent.

Alcohol helps to prevent super saturation.

Limit test forIron

Principle:

Limit test of Iron is based on the reaction of iron in ammonical solution with thioglycollic acid in presence of citric acid to form iron thioglycolate which is pale pink to deep reddish purple in colour.

Procedure:

Test sample	Standard compound
Sample is dissolved in specific amount of water and then volume is made up to 40 ml	2 ml of standard solution of iron diluted with water upto 40ml
Add 2 ml of 20 % w/v of citric acid (iron free)	Add 2 ml of 20 % w/v of citric acid (iron free)
Add 2 drops of thioglycollic acid	Add 2 drops of thioglycollic acid
Add ammonia to make the solution alkaline and adjust the volume to 50 ml	Add ammonia to make the solution alkaline and adjust the volume to 50 ml
Keep aside for 5 min	Keep aside for 5 min
Color developed is viewed vertically and compared with standard solution	Color developed is viewed vertically and compared with standard solution

Earlier aamoniumthiocyanate reagent was used for the limit test of iron. Since thioglycolic acid is more sensitive reagent, it has replaced ammonium thiocyanate in the test.

Observation:

The purple color produce in sample solution should not be greater than standard solution. If purple color produces in sample solution is less than the standard solution, the sample will pass the limit test of iron and vice versa.

Reasons:

Citric acid helps precipitation of iron by ammonia by forming a complex with it.

Limit test for Heavy Metals

Principle:

Limit test of heavy metals is based on the reaction of metallic impurities with hydrogen sulfide in acidic medium to form brownish colour solution. Metals that responseto this test are lead, mercury, bismuth, arsenic, antimony, tin, cadmium, silver, copper, and molybdenum.

The metallic impurities in substances are expressed as parts of lead per million parts of the substance. The usual limit as per Indian Pharmacopoeia is 20ppm

Procedure:

The Indian Pharmacopoeia has adopted three methods for the limit test of heavy metals.

Method I: Use for the substance which gives clear colorless solution under the specific condition.

Test sample	Standard compound
Solution is prepared as per the monograph and 25 ml is transferred in Nessler’s cylinder	Take 2 ml of standard lead solution and dilute to 25 ml with water
Adjust the pH between 3 to 4 by adding dilute acetic acid ‘Sp’ or dilute ammonia solution ‘Sp’	Adjust the pH between 3 to 4 by adding dilute acetic acid ‘Sp’ or dilute ammonia solution ‘Sp’
Dilute with water to 35 ml	Dilute with water to 35 ml
Add freshly prepared 10 ml of hydrogen sulphide solution	Add freshly prepared 10 ml of hydrogen sulphide solution
Dilute with water to 50 ml	Dilute with water to 50 ml
Allow to stand for five minutes	Allow to stand for five minutes
View downwards over a white surface	View downwards over a white surface

Observation:

Thecolor produce in sample solution should not be greater than standard solution. If color produces in sample solution is less than the standard solution, the sample will pass the limit test of heavy metals and vice versa.

Limit test for Lead

Lead is a most undesirable impurity in medical compounds and comes through use of sulphuric acid, lead lined apparatus and glass bottles use for storage of chemicals.

Principle:

Limit test of lead is based on the reaction of lead and diphenylthiocabazone (dithizone) in alkaline solution to form lead dithizone complex which is read in color.

Dithizone is green in color in chloroform and lead-dithizone complex is violet in color, so the resulting color at the end of process is red.

Procedure:

Test sample	Standard compound
A known quantity of sample solution is transferred in a separating funnel	A standard lead solution is prepared equivalent to the amount of lead permitted in the sample under examination
Add 6ml of ammonium citrate	Add 6ml of ammonium citrate
Add 2 ml of potassium cyanide and 2 ml of hydroxylamine hydrochloride	Add 2 ml of potassium cyanide and 2 ml of hydroxylamine hydrochloride
Add 2 drops of phenol red	Add 2 drops of phenol red
Make solution alkaline by adding ammonia solution.	Make solution alkaline by adding ammonia solution.
Extract with 5 ml of dithizone until it becomes green	Extract with 5 ml of dithizone until it becomes green
Combine dithizone extracts are shaken for 30 mins with 30 ml of nitric acid and the chloroform layer is discarded	Combine dithizone extracts are shaken for 30 mins with 30 ml of nitric acid and the chloroform layer is discarded
To the acid solution add 5 ml of standard dithizone solution	To the acid solution add 5 ml of standard dithizone solution
Add 4 ml of ammonium cyanide	Add 4 ml of ammonium cyanide
Shake for 30 mins	Shake for 30 mins
Observe the color	Observe the color