Limit Test

Limit test is defined as quantitative or semi quantitative test designed to identify and control small quantities 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 but to identify the impurities present in the substance and compare it with standard. 

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

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 soluble chloride with silver nitrate in presence of dilute nitric acid to form silver chloride, which appears as solid particles (Opalescence) in the solution.

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 the limit test of chloride and visa versa.

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 the solution.

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

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

Observation:

The turbidity 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 the test by giving ionic concentration in the reagent. 

Alcohol helps to prevent super saturation.

Limit test for Iron

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 response to 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 20 ppm

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:

 

The color 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

Observation:

 

The intensity of the color of complex, is depends on the amount of lead in the solution. 

The color  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 lead and vice versa.

 

Reasons:

Ammonium citrate, potassium cyanide, hydroxylamine hydrochloride is used to make pH

optimum so interference and influence of other impurities have been eliminated.

Pharmaceutical Impurities and Limit Test Notes PDF