Principle: The limit test for heavy metals is designed to determine the content of metallic impurities that are coloured by hydrogen sulphide or sodium sulphide under the condition of the test. The heavy metals (metallic impurities) may be iron, copper, lead, nickel, cobalt, bismuth, antimony etc. The limit for heavy metals is indicated in the individual monograph in terms of ppm of lead i.e. the parts of lead per million parts of the substance being examined. The Indian pharmacopoeia had adopted four methods (methods A, B, C and D) for the limit test for heavy metals.
Note: Methods A, B and C are based upon the reaction of the heavy metal ion with hydrogen sulphide (in methods A and B) or sodium sulphide (in method C) leading to the formation of heavy metal sulphides. The metal sulphides remain distributed in a colloidal state and give rise to brownish colouration. The colour produced in the test solution is compared with that of the standard solution containing a definite amount of lead.
Method D is based upon the precipitation of relatively insoluble and characteristically coloured sulphides of heavy metals when aqueous solutions are treated with alkali metal sulphides (NaSH). NaSH is generated immediately before use by heating thioacetamide with sodium hydroxide solution. In this test, there is a formation of brown colour because of the precipitation of metal sulphides at about pH 3.5 in colloidal form which is stabilized by the glycerin. The colour is compared by keeping the two Nessler cylinders side by side and viewing vertically downwards against a white background. The usual limit for heavy metals as per I.P. is 20 ppm.
Reaction:
Heavy metals + H2S/Na2S → Heavy metal sulphides (Brownish colouration)
Preparation of Reagent/Solution:
Standard lead solution (20 ppm Pb): Dilute 1 volume of standard lead solution (0.1% Pb) to 50 volumes with water. Procedure:
METHOD (I.P. 1996)
Method (A):
It is applicable for the substance, which gives a clear colourless solution under specified conditions of the test.
Standard solution: Pipette 1.0 mL of standard lead solution (20 ppm Pb) into a Nessler cylinder labelled as “Standard” and dilute to 25.0 mL with water. Adjust the pH between 3.0 and 4.0 with dilute acetic acid or dilute ammonia solutions, dilute to 35.0 mL with water and mix.
Test solution: Take 25.0 mL of the solution prepared as directed in the individual monograph into a Nessler cylinder labelled as “Test” or dissolve the specified quantity of the substance in water to produce 25.0 mL. Adjust the pH between 3.0 and 4.0 with dilute acetic acid or dilute ammonia solution, dilute to 35.0 mL with water and mix.
Procedure: Add 10.0 mL of freshly prepared hydrogen sulphide to each of the Nessler cylinders containing the test solution and the standard solution respectively. Mix, dilute to 50.0 mL with water and allow to stand for 5 minutes. Compare the colour by viewing vertically downwards over a white surface. The colour produced with the test solution is not more intense than that produced with the standard solution.
| Sr.No. | Test | Standard |
| 1 | Place 25 mL of a solution of the test sample in a 50 mL Nessler cylinder. | Place 2 mL of standard lead solution (20 ppm Pb). |
| 2 | Adjust the pH between 3.0 and 4.0 with dilute acetic acid or dilute ammonia solution. | Adjust the pH between 3.0 and 4.0 with dilute acetic acid or dilute ammonia solution. |
| 3 | Dilute to 35.0 mL with water and mix. | Dilute to 35.0 mL with water and mix. |
| 4 | Add 10.0 mL of freshly prepared hydrogen sulphide. | Add 10.0 mL of freshly prepared hydrogen sulphide. |
| 5 | Mix, dilute to 50.0 mL with water and allow to stand for 5 minutes. | Mix, dilute to 50.0 mL with water and allow to stand for 5 minutes. |
Interpretation: Compare the intensity of colour in the two Nessler cylinders by viewing vertically downwards. For passing the test, the intensity of colour produced in the test sample should not be more than that of the standard.
Method (B):
It is applicable for those substances, which do not give a clear colourless solution under the specified conditions.
Standard solution: (Same as for Method – (A))
Test solution: Weigh the quantity of the substance as specified in the individual monograph in a crucible, wet the sample by adding sufficient sulphuric acid, and ignite at low temperature to thorough charring. To the charred mass add 2.0 mL of nitric acid and 5 drops of sulphuric acid and heat until white fumes are no longer evolved. Ignite at 500°C to 600°C until the carbon is completely burnt off. Cool, add 4.0 mL of dilute hydrochloric acid, digest in a water bath for 15 minutes and evaporate to dryness in a water bath. Moisten the residue with 1 drop of hydrochloric acid, add 10.0 mL of hot water and digest for 2 minutes. Make the solution just alkaline to the litmus paper by dropwise addition of ammonia solution. Dilute to 25.0 mL with water and adjust the pH between 3.0 and 4.0 with dilute acetic acid. Filter, rinse the crucible and filter with 10.0 mL of water. Combine the filtrate and wash in a Nessler cylinder labelled as “Test”, dilute to 35.0 mL with water and mix.
Procedure: (Same as for Method A)
Method (C):
It is applicable to those substances that give a clear colourless solution in sodium hydroxide. Standard solution: Pipette 1.0 mL of standard lead solution (20 ppm Pb) into a Nessler cylinder labelled as “Standard”, add 5.0mL of dilute sodium hydroxide solution, dilute to 50.0mL with water and mix.
Test solution: Place 25.0 mL of the solution prepared, as directed in the individual monograph into a Nessler cylinder labelled as ‘Test’ or dissolve the specified quantity of the substance in 20 mL of water and 5.0 mL of dilute sodium hydroxide solution. Dilute to 50.0 mL with water and mix.
Procedure: Add 5 drops of sodium sulphide solution to each of the Nessler cylinders containing the standard solution and the test solution respectively. Mix and allow to stand for 5 minutes and compare the colour by viewing vertically downwards over a white surface. The colour produced with the test solution is not more intense than that produced with the standard solution.
| Sr. No | Test | Standard |
| 1 | Place 25 mL of a solution of the test sample in a 50 mL Nessler cylinder. | Place 2 mL of standard lead solution (20 ppm Pb). |
| 2 | Add 5 ml of dilute NaOH solution. | Add 5 ml of dilute NaOH solution. |
| 3 | Add distilled water up to 50 mL and mix. | Add distilled water up to 50 mL and mix. |
| 4 | Add 5 drops of sodium sulphide solution and stir. | Add 5 drops of sodium sulphide solution and stir. |
| 5 | Allow to stand for 5 minutes. | Allow to stand for 5 minutes. |
Interpretation: Compare the intensity of colour in the two Nessler cylinders by viewing vertically downwards against a white background. For passing the test, the intensity of colour produced in the test sample should not be more than that of the standard.
Method (D):
Standard solution: Pipette 10.0 mL of either standard lead solution (1 ppm Pb) or standard lead solution (2 ppm Pb) into a small Nessler cylinder labelled as “Standard”. Add 2.0 mL of the test solution and mix.
Test solution: Prepare as directed in the individual monograph and pipette 12.0 mL into a small Nessler cylinder labelled as “Test”.
Preparation of Reagents/Solutions:
Standard lead solution (2 ppm Pb): Dilute 1 volume of standard lead solution (20 ppm Pb) to 10 volumes with water.
Thioacetamide reagent: To 0.2 mL of 4% w/v thioacetamide solution, add 1.0 mL of a mixture of 15.0 mL of 1M sodium hydroxide, 5.0 mL of water and 20.0 mL glycerin (85%). Heat on a water bath for 20 seconds, cool and use immediately.
Acetate buffer (pH 3.5): Dissolve 25 g of ammonium acetate in 25.0 mL of water and add 38.0 mL of 7 M hydrochloric acid. Adjust the pH to 3.5 by using 3 M hydrochloric acid or 6 M ammonia and dilute to 100.0 mL with water.
Procedure: Add 2.0 mL of acetate buffer (pH 3.5) to each of the above Nessler cylinders, mix, add 1.2 mL of thioacetamide reagent and allow to stand for 2 minutes. Compare the colour by viewing vertically downwards over a white surface. The colour produced with the test solution is not more intense than that produced with the standard solution.
| Sr.No | Test | Standard |
| 1 | Place 12 mL of a solution of the test sample in a 50 mL Nessler cylinder. | Place 10.0 mL of standard lead solution (1 ppm Pb), Add 2.0 mL of the test solution and mix. |
| 2 | Add 2.0 mL of acetate buffer pH 3.5 and mix. | Add 2.0 mL of acetate buffer pH 3.5 and mix. |
| 3 | Add 1.2 mL of thioacetamide reagent. | Add 1.2 mL of thioacetamide reagent. |
| 4 | Add distilled water up to 50 mL and mix. | Add distilled water up to 50 mL and mix. |
| 5 | Allow to stand for 2 minutes. | Allow to stand for 2 minutes. |
Interpretation: Compare the intensity of colour in the two Nessler cylinders by viewing vertically downwards against a white background. For passing the test, the intensity of colour produced in the test sample should not be more than that of the standard.
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