Limit Test For Heavy Metals

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 color 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.

Table of Contents

Procedure
The Indian Pharmacopoeia has adopted three methods for the limit test of heavy metals. As follows,

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

Method II: Use for the substance which do not give clear colorless solution under the specific condition.

Method III: Use for the substance which gives clear colorless solution in sodium hydroxide solution.

Method I

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 or dilute ammonia solution	Adjust the pH between 3 to 4 by adding dilute acetic acid or dilute ammonia solution
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.

Method II

Test sample	Standard compound
Weigh specific quantity of test substance, moisten with sulphuric acid and ignite on a low flame till completely charred Add few drops of nitric acid and heat to 500 °C Allow to cool and add 4 ml of hydrochloric acid and evaporate to dryness Moisten the residue with 10 ml of hydrochloric acid and digest for two minutes Neutralize with ammonia solution and make just acid with acetic acid	Take 2 ml of standard lead solution and dilute to 25 ml with water
Adjust the pH between 3 to 4 and filter if necessary	Adjust the pH between 3 to 4 by adding dilute acetic acid or dilute ammonia solution
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

Method III

Test sample	Standard compound
Solution is prepared as per the monograph and 25 ml is transferred in Nessler’s cylinder or weigh specific amount of substance and dissolve in 20 ml of water and add 5 ml of dilute sodium hydroxide solution	Take 2 ml of standard lead solution
Make up the volume to 50 ml with water	Add 5 ml of dilute sodium hydroxide solution and make up the volume to 50 ml with water
Add 5 drops of sodium sulphide solution	Add 5 drops of sodium sulphide solution
Mix and set aside for 5 min	Mix and set aside for 5 min
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. Phenol red is used as indicator to develop the color at the end of process. Lead present as an impurities in the substance, gets separated by extracting an alkaline solution with a dithizone extraction solution.

Limit test for Arsenic

Principle
Limit test of Arsenic is based on the reaction of arsenic gas with hydrogen ion to form yellow stain on mercuric chloride paper in presence of reducing agents like potassium iodide. It is also called as Gutzeit test and requires special apparatus.

Arsenic, present as arsenic acid in the sample is reduced to arsenious acid by reducing agents like potassium iodide, stannous acid, zinc, hydrochloric acid, etc. Arsenious acid is further reduced to arsine (gas) by hydrogen and reacts with mercuric chloride paper to give a yellow stain.

H₃AsO₄ + H₂SnO₂ → H₃AsO₃ + H₂SnO₃
Arsenic acid Arsenious acid

H₃AsO₃ + 3H₂ → AsH₃ +3H₂O
Arsenious acid Arsine

The depth of yellow stain on mercuric chloride paper will depend upon the quality of arsenic present in the sample.

Procedure

Test solution

The test solution is prepared by dissolving specific amount in water and stagnated HCl (arsenic free) and kept in a wide mouthed bottle.
To this solution 1 gm of KI, 5 ml of stannous chloride acid solution and 10 gm of zinc is added (all these reagents must be arsenic free).
Keep the solution aside for 40 min and stain obtained on mercuric chloride paper is compared with standard solution.

Standard solution

A known quantity of dilute arsenic solution is kept in wide mouthed bottle and rest procedure is followed as described in test solution (using Gutzeit apparatus)

Reasons
Stannous chloride is used for complete evolution of arsine Zinc, potassium iodide and stannous chloride is used as a reducing agent. Hydrochloric acid is used to make the solution acidic, Lead acetate pledger or papers are used to trap any hydrogen sulphide which may be evolved along with arsine.