INTRODUCTION:

In Chemistry, Limit means a value or amount that is likely to be present in a substance and test means to examine or to investigate. Thus, limit test is nothing but to identify the impurities in the substance and compare it with standard. In general, 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 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. Limit test of sulphate is based on the reaction of soluble sulphate with barium chloride in presence of alcohol and potassium sulphate to form barium sulphate, which appears as solid particles (turbidity) in the solution. Here alcohol is added to prevent super saturation. Limit test of heavy metals is based on the reaction of metallic impurities with hydrogen sulfide in acidic medium to form colored solution. Metals that response to this test are lead, mercury, bismuth, arsenic, antimony, tin, cadmium, silver, copper, and molybdenum. Limit test of lead is based on the reaction of lead and diphenyl thiocabazone (dithizone) in alkaline solution to form lead dithizone complex which is read in color. Limit test of Iron is based on the reaction of iron in ammonical solution with thioglycollic acid to form iron thioglycolate which is pink-reddish purple in color. 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. ^[1-3]

VARIOUS LIMIT TEST

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:
TABLE :1

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.^[3-5]

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:
TABLE :2

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. ^[5-7]

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 color.

Procedure:

TABLE :3

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.

Thioglycolic acid helps to oxidize iron (II) to iron (III).

Ammonia to make solution alkaline. ^[7-9]

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.

TABLE :4

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. ^[7-9]

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

TABLE :5

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 ‘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:

TABLE :6

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.^[9-10]

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:

TABLE :6

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 bye 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 stannated 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 this 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 (Gutzeit apparatus shown in Fig.1).

Fig.1 Gutzeit apparatus.

A ： approximately 60 ml generator bottle with 40 ml indicating line.

B ： glass tube with 6.5 mm inner diameter

C and D ： a ground joint glass tube with 6.5 mm inner diameter and 18 mm outer diameter at the joint. Inner joint and the outer joint form a concentric circle.

E ： rubber stopper

F ： narrow part of the glass tube B. Glass wool is inserted up to this part.

G ： rubber board (Lead acetate cotton plug)

H ： clamp

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.^[2-5]

REFFERENCE

1. A.H. Beckett and J.B. Stenlake, Practical pharmaceutical chemistry, Part-I. The Athtone press, University of London, London.

2. P. Gundu Rao, Inorganic Pharmaceutical Chemistry; Vallabh Prakashan, Delhi.

3. Advanced Inorganic Chemistry by Satya Prakash, G.D. Tuli

4. Jolly-Modern Inorganic Chemistry

5. Pharmaceutical Inorganic Chemistry Textbook by Alagarsamy.

6. L.M. Atherden, Bentley and Driver’s Textbook of Pharmaceutical Chemistry Oxford University Press, London.

7. Indian Pharmacopoeia 1996, 2006.

8. J.H Block, E. Roche, T.O Soine and C.O. Wilson, Inorganic Medical and Pharmaceutical Chemistry Lea & Febiger Philadelphia PA.

9. Pharmaceutical Inorganic Chemistry by S. Chand, R.D. Madan, Anita Madan

10. Pharmaceutical Inorganic Chemistry by Soma Shekar Rao

Received on 08.12.2016 Accepted on 08.01.2017

Asian J. Pharm. Res. 2017; 7(1): 30-34.

DOI: 10.5958/2231-5691.2017.00006.5