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Limit Test for Arsenic

Limit Test for Arsenic and Heavy Metals

Alok Bains

10/6/20248 min read

Limit Test for Arsenic

  • Arsenic is a very toxic element and cumulative in nature. Even trace arsenic in pharmacopoeial substances is harmful for the human body. Indian pharmacopeia prescribes limits of arsenic impurity present in the pharmacopoeial compounds. For example, ot should not be more than 1 PPM in sodium chloride and not more than 2 PPM in sodium citrate.

  • Gutzeit apparatus (Apparatus for Arsenic limit test) is used for limit test for arsenic. This is a modified method Gutzeit test. Thus it is also called the modified Gutzeit test. Separate guitzeit apparatus is used for “Standard” and “Test” samples. It consists of following parts:

    • 1. Wide mouth bottle: It consists of a wide wide-mouthed glass bottle capacity of 12 ml. Its mouth diameter is about 2.5 cm. Its mouth is closed by using rubber bung.

    • 2. A glass tube: A 200 mm (20 cm) long glass tube having an outer diameter of 8 mm and inner diameter of 6.5 mm enters the glass bottle through a rubber bung. One end of the glass tube outside the bottle is open end. Its end inside the bottle is narrow with a 1 mm diameter. A 2 mm hole is present just above the narrow end of the glass tube. This hole allows slow entry of arsenic gas from the bottle into the glass tube during the arsenic test procedure. Actually, it is an alternative passage for arsenic gas into the glass tube if the narrow end is blocked by water condensation.

    • 3. Acetate cotton wool: The glass tube is lightly packed with cotton wool previously moistened with lead acetate solution.

    • 4. Rubber bungs: Two rubber bungs with concentric holes are attached to the open end of the glass tube outside the glass bottle. Both bungs are fixed together by using clips so that concentric holes of both bungs make a true tube inside both bungs.

    • 5. Mercuric chloride paper (HgCl2): Dry mercuric chloride paper is placed flat at the top of the lower rubber bung and below the upper rubber bung. Both rubber bungs are clipped so that both rubber bung tubes are concentric. Thus, Dry mercuric chloride paper is present in between both rubber bungs.

  • Principle: Pharmacopoeia prescribes a quantitative test for the arsenic limit test in pharmacopoeial substances.

    • 1. Conversion of Arsenic to Arsenic Acids: The sample or aqueous extract of the sample is dissolved in the acid. Acid converts arsenic present in the sample solution to arsenic acid or arsenious acid. This depends upon the valency of arsenic impurity present in the pharmacopoeial substances. Trivalent arsenic produces arsenic acid. Pentavalent arsenic produces arsenic acid.

    • 2. Arsenic Reduction: The sample solution is treated with a reducing agent such as stannous chloride or sulfurous acid to convert pentavalent arsenic acid to trivalent arsenious acid. IP/BP recommends using stannous chloride. USP recommends using sulfurous acid in place of stannous chloride.

    • 3. Arsenic Detection using nascent hydrogen: Nascent hydrogen is used to convert arsenic acid to arsine gas. Arsine gas reacts with mercuric chloride paper to develop a yellow or brown stain on mercuric chloride paper. The intensity of color depends upon arsenic concentration in the pharmacopoeial substance.

    • 4. Temperature: The rate of reaction is very slow at room temperature. Thus, the temperature is raised and maintained at 40 degrees C by using a water bath.

    • 5. Result: The color intensity of the test sample less than the standard sample indicates pharmacopoeial substance passes the limit test for Arsenic.

  • 6. Explanation: Hydrogen sulfide (H2S) is released during a chemical reaction. It reacts with mercuric chloride paper present in between two bungs. This develops stains on mercuric chloride paper. That will interfere with the result. Thus, cotton wool moistened with lead acetate solution is placed inside the glass tube of the Gutzeit apparatus. Lead acetate absorbs the Hydrogen sulfide (H2S) and is released during a chemical reaction inside a glass bottle.

  • Reagents: All reagents must be free from arsenic impurity.

    • 1. Stannous chloride AsT solution in hydrochloric acid as specified in the pharmacopeia.

    • 2. Potassium iodide AsT: 1M Potassium iodide solution and

    • 3. 10 gm granulated zinc AsT. Zinc is used to produce nascent hydrogen. Granulated zinc releases nascent hydrogen at a steady speed for a prolonged duration compared to ordinary zinc.

    • 4. Arsenic standard solution: It contains 1.32 mg arsenic trioxide in 100 ml distilled water (It is equivalent to 1 mg arsenic in 100 ml).

  • Explanation:

    • 1. AsT means ragent free from arsenic impurity.

    • 2. Hydrochloric acid is used to convert arsenic present in the sample solution to arsenic acid or arsenious acid.

    • 3. Stannous chloride AsT is used as a reducing agent to convert pentavalent arseinic acid to trivalent arseneous acid.

    • 4. Potassium iodide (KI) forms hydroiodic acid (HI) in reaction. Hydroiodic acid helps in the reduction of pentavalent arsenic acid to trivalent arsenic acid.

  • Procedure:

    • A. Test solution in Gutzeit apparatus:

      • · Place 50 ml distilled water in the Gutzeit apparatus (Arsenic test apparatus). Label it as (Test).

      • · Add sample as the amount specified in pharmacopeia into the arsenic test apparatus.

      • · Add 10 ml stannous chloride solution in hydrochloric acid into the arsenic test apparatus.

      • · Add 10 gm of granulated zinc into the arsenic test apparatus.

      • · Fit the bung containing glass tube in the mouth of the arsenic test apparatus bottle quickly.

      • · Allow the reaction to proceed for 40 minutes at a temperature of 40 degrees C. Temperature is maintained by using a water bath.

    • B. Test solution in Gutzeit apparatus:

      • · Place 50 ml distilled water in the Gutzeit apparatus (Arsenic test apparatus). Label it as (Test).

      • · Add 1 ml arsenic standard solution (10 PPM As) to the arsenic test apparatus.

      • · Add 10 ml stannous chloride solution in hydrochloric acid into the arsenic test apparatus.

      • · Add 10 gm of granulated zinc into the arsenic test apparatus.

      • · Fit the bung containing glass tube into the mouth of the arsenic test apparatus bottle quickly.

      • · Allow the reaction to proceed for 40 minutes at a temperature of 40 degrees C. Temperature is maintained by using a water bath.

  • Observation/Interpretation: Compare the yellow stain developed on the mercuric chloride paper in daylight without any delay. Mercuric chloride paper test stain intensity should not be more than the stain intensity on the Mercuric chloride paper standard. Arsenic impurity is reported in PPM. One PPM means 1 mg in 1 kg OR 1 mcg in 1 gram. Pharmacopoeia prescribes an arsenic impurity limit of not more than 4 PPM.

Alok Bains

Limit Test For Heavy Metals

Heavy metals are a group of naturally occurring elements that have higher molecular weight and density than water. Examples of heavy metals in pharmacoeial substances as impurities are lead, tin, cobalt, manganese, arsenic, zinc, cadmium, silver, copper, molybdenum, etc. Pharmacopoeia prescribes limits for these heavy metals in pharmacopoeial substances. The heavy metals impurities in pharmacopoeial substances are expressed as parts of lead per million parts of the substance. The Indian Pharmacopoeia prescribes this limit not more than 20 ppm.

Indian Pharmacopoeia prescribes three methods for limit test of heavy metals:

  • 1. Method-I: It is for pharmacopeial substances that form colorless solutions.

  • 2. Method-II: It is for pharmacopoeial substances that do not form a clear colorless solution

  • 3. Method-III: It is used for pharmacopoeial substances that form colorless solutions in the presence of sodium hydroxide solution.

Principle: Heavy metals react with hydrogen sulfide solution in acidic media to form metal sulfides. Metal sulfides develop a brownish-color colloid solution. Freshly prepared saturated solution of hydrogen sulfide is used in limit tests for heavy metals.

  • Heavy metal + H2S = Heavy metal sulfides (Brown color colloid)

  • In the limit test for heavy metals, Acetic acid (10% V/V) or Dilute ammonia solution (10% V/V) is used to adjust pH 3.0 to 4.0. This provides acidic media for reaction between heavy metals and hydrogen sulfide.

  • A lead solution containing 0.1598 gm lead nitrate and, 1ml concentrated hydrochloric acid in quantity sufficient purified water to produce 1000 ml volume is used as a standard lead solution.

Method-I: It is for pharmacopeial substances that form colorless solutions.

Procedure

  • 1. Test solution

    • Prepare the solution of the test sample as prescribed in the monograph of pharmacopeia.

    • Place 25 ml of the test sample solution in a Nessler cylinder labeled “Test”

    • Adjust the pH of the solution between 3.0 and 4.0 by using dilute acetic acid or ammonia solution

    • Dilute it with distilled water up to 35 ml

    • Add 10 ml of freshly prepared hydrogen sulfide solution

    • Add sufficient distilled water to make a volume of 50 ml.

    • Stir using a glass rod and set aside for 5 minutes

  • 2. Standard solution

    • Prepare a solution of standard lead solution as prescribed in pharmacopeia.

    • Place 2 ml of standard lead solution in a Nessler cylinder labeled “Standard”

    • Adjust the pH of the solution between 3.0 and 4.0 by using dilute acetic acid or ammonia solution

    • Dilute it with distilled water up to 35 ml

    • Add 10 ml of freshly prepared hydrogen sulfide solution

    • Add sufficient distilled water to make a volume of 50 ml.

    • Stir using a glass rod and set aside for 5 minutes

  • Observation/Interpretation: Place both test and standard Nessller cylinder on a white surface. View Nessler cylinder vertically downward. Compare the color of both Nessler cylinders. Color in the Test Nessler cylinder should not be more intense than color in the Standard Nessler cylinder.

Method II: It is for pharmacopoeial substances that do not form a clear colorless solution.

Procedure

  • 1. Test solution

    • Prepare the solution of the test sample as prescribed in the monograph of pharmacopeia.

    • Place 25 ml of the test sample solution in a Nessler cylinder labeled “Test”

    • Adjust the pH of the solution between 3.0 and 4.0 by using dilute acetic acid or ammonia solution

    • Dilute it with distilled water up to 35 ml

    • Add 10 ml of freshly prepared hydrogen sulfide solution

    • Add sufficient distilled water to make a volume of 50 ml.

    • Stir using a glass rod and set aside for 5 minutes

  • 2. Standard solution

    • Prepare a solution of standard lead solution as prescribed in pharmacopeia.

    • Place 2 ml of standard lead solution in a Nessler cylinder labeled “Standard”

    • Adjust the pH of the solution between 3.0 and 4.0 by using dilute acetic acid or ammonia solution

    • Dilute it with distilled water up to 35 ml

    • Add 10 ml of freshly prepared hydrogen sulfide solution

    • Add sufficient distilled water to make the volume of 50 ml.

    • Stir using a glass rod and set aside for 5 minutes

  • Observation/Interpretation: Place both test and standard Nessller cylinder on a white surface. View Nessler cylinder vertically downward. Compare the color of both Nessler cylinders. Color in the Test Nessler cylinder should not be more intense than color in the Standard Nessler cylinder.

Method III: It is used for pharmacopoeial substances that form colorless solutions in the presence of sodium hydroxide solution.

Procedure

  • 1. Test solution

    • Prepare the solution of the test sample as prescribed in the monograph of pharmacopeia.

    • Place 25 ml of the test sample solution in a Nessler cylinder labeled “Test”

    • Add 5 ml of dilute sodium hydroxide solution

    • Add distilled water to make a volume of 50 ml.

    • Add 5 drops of sodium hydroxide solution

    • Stir using a glass rod and set aside for 5 minutes

  • 2. Standard solution

    • Prepare a solution of standard lead solution as prescribed in pharmacopeia.

    • Place 2 ml of standard lead solution in a Nessler cylinder labeled “Standard”

    • Add 5 ml of dilute sodium hydroxide solution

    • Add distilled water to make a volume of 50 ml.

    • Add 5 drops of sodium hydroxide solution

    • Stir using a glass rod and set aside for 5 minutes

  • Observation/Interpretation: Place both test and standard Nessller cylinder on a white surface. View Nessler cylinder vertically downward. Compare the color of both Nessler cylinders. Color in the Test Nessler cylinder should not be more intense than color in the Standard Nessler cylinder.

Alok Bains