Limit Test

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LIMIT TEST

·         Limit tests are quantitative or semi-quantitative test design to identify and control small quantities of impurities which are likely to be present in the substance.

·         Tests being used to identify impurities.

·         Tests being used to control the impurities.

·         All the limit test that are prescribed in the pharmacopoeias are based on the comparison of standard turbidity or colour which that of the sample under test.

·         Limit tests prescribed in parts per millions (PPM).

TYPES

1.       Test in which there is no visible reaction

·         There is no colour, opalescence or precipitate → negative test indicates the absence of large impurity.

2.       Comparison method

·         Compare the amount of impurity in the substance with a standard of known concentration and determine whether impurity is within or the excess of the limit prescribe.

3.       Quantitative determination

·          Amount of impurities present in actually determined and compare with the numerical limit given in the pharmacopoeia.

FACTORS AFFECTING LIMIT TEST

·         Specificity of the tests

·         Sensitivity

·         Control of personal error (Analyst error)

o    Test in which there is no visible reaction.

o    Comparison methods

o    Quantitative determination

LIMIT TEST FOR SULPHATE

·         The sulphate limit test is designed to determine the allowable limit of sulfate contain in a sample.

Principle

 

·         Limit test of sulphate is based on the reaction of soluble sulphate with barium chloride in presence of dilute HCl to form barium sulphate which appears as solid particle (turbidity) in the solution.

·         Then the comparison of turbidity is done with a standard turbidity form a known amount if sulphate and same volume of dilute HCl have been added to both solutions.

·         The barium chloride reagent test solution in the IP has been replaced by barium sulphate reagent which is having barium chloride, sulphate free alcohol and a solution of potassium sulphate has been added to increase the sensitivity of the test.

 Procedure

Test

Standard

·         Specific weight of compound is dissolved in water or solution is prepared as directed in the pharmacopoeia and transferred in the Nessler cylinder.

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·         Add 2 ml of dilute HCl.

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·         Dilute to 45 ml in Nessler cylinder.

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·         Add 5 ml of barium sulphate reagent.

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·         Keep aside for 5 minutes.

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·         Observe the turbidity.

·         Take 1 ml of 0.1089% w/v solution of potassium sulphate in Nessler cylinder.

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·         Add 2 ml of dilute HCl.

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·         Dilute to 45 ml in Nessler cylinder.

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·         Add 5 ml of barium sulphate reagent.

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·         Keep aside for 5 minutes.

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·         Observe the turbidity.

 Reasons

·         The turbidity produce in sample solution should not be greater than the standard solution. Less turbidity than standard pass the limit test and vice versa.

LIMIT TEST FOR IRON

·         Limit test of Iron is based on the principle on the reaction of Iron in ammonical solution with thioglycolic acid in presence of citric acid to form Iron thioglycolate (Ferrous thioglycolate complex) which produces pale pink to deep radish purple color in alkaline medium.

·         Thioglycolic acid is used as reducing agent.

·         The colour of the ferrous thioglycolate complex fades in the presence of air due to oxidation.

·         Also, the colour is destroyed in presence of oxidizing agents and strong alkalis.

·         The purple colour is developed only in alkaline medium so ammonia solution is used. But ammonia reacts with Iron forms precipitate of ferrous hydroxide.

·         Thus, citric acid is used which prevents the precipitate of Iron with ammonia by forming a complex with Iron as Iron citrate.

Procedure

Test

Standard

·         Sample is dissolved is specific amount of water and then volume is made up to 40 ml.

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·         Add 2 ml of 20% w/v of citric acid (Iron free).

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·         Add 2 drops of thioglycolic acid.

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·         Add ammonia to make the solution alkaline and adjust the volume to 50 ml.

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·         Keep aside for 5 min.

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·         Colour developed is viewed vertically and compared with standard solution.

·         2 ml of standard solution of Iron diluted with water up to 40 ml.

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·         Add 2 ml of 20% w/v of citric acid (Iron free).

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·         Add 2 drops of thioglycolic acid.

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·         Add ammonia to make the solution alkaline and adjust the volume to 50 ml.

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·         Keep aside for 5 min.

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·         Colour developed is viewed vertically and compared with standard solution.

 Observation

·         The purple colour in the sample solution should not be greater than standard solution. If the purple colour produces in the sample solution is less than the standard solution, the sample will pass the limit test of Iron and vice versa.

Reason

·         Citric acid forms complex with metal cation and helps precipitation of Iron by forming a complex with it.

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

·         Ammonia is added to make solution alkaline. The pale pink colour is visible only in the alkaline media.

·         Colour is not visible in acid media as ferrous thioglycolate complex decomposes in high acidic media.

LIMIT TEST FOR ARSENIC

·         Arsenic is a well-known undesirable and harmful impurity, which is present in medicinal substances.

·         All pharmacopoeia prescribes a limit test for it.

·         Also known as Gutzeit test.

·         All the special reagents used in the limit test for arsenic are marked and distinguished by letter “T”, which indicates all should be Arsenic free and should themselves conform to the 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 agent like potassium iodide.

·         Arsenic, present as arsenic acid (H2ASO4) in the sample is reduced to arsenious acid (H2ASO3) by reducing agent like potassium iodide, stannous acid, zinc, H2SO4 etc.

 

·         Arsenious acid is further reduced to arsenic gas (ASH3) by hydrogen and reacts with mercuric chloride paper to give YELLOW STAIN.

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

·         When the sample dissolved in acid, the arsenic present in the sample gets converted to arsenic acid.

·         By the action of reducing agents like KI, stannous acid etc. arsenic acid gets reduced to arsenious acid.

·         The nascent hydrogen formed during the reaction, which further reduce arsenious acid to arsine gas, which reacts with HgCl2 paper, gives yellow stain. 

Apparatus

·         It is having a wide mouthed glass bottle of 120 ml capacity having mouth of about 2.5 cm in diameter, this bottle is fitted with a rubber bung through which passes a glass tube, 20 com long.

·         External diameter → 0.8 cm.

·         Internal diameter → 0.65 cm

·         The tube is constricted at its lower end extremity to about 1mm in diameter and there is blown hole, not less than 2 mm in diameter, in the side of the tube near the constricted part.

·         The upper end of the glass is fitted with two rubber (25mm × 25mm), each having a hole bored centrally and exactly 6.5 mm in diameter.

·         One of the bungs has been fitted to the upper end of the tube, while second bung has to be fitted upon the first bung in such a way that the mercury chloride paper gets exactly sandwich between the central perforation of the two.

·         The bungs are kept in close contact by using rubber band or spring dip in such a manner that the gas evolved from the bottle must have to pass through the 0.65 mm internal circle of mercuric chloride paper.

 

·         During the test, the evolved gases have been passing through the side hole, the lower hole serving as an exit for water which condenses in the constricted part of the tube.

Procedure

Test

Standard

·         The test solution is prepared by dissolving specific amount of water and stannated HCl (arsenic free) and kept in a wide mouthed bottle.

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·         Add 1 gm of KI.

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·         5 ml of stannous chloride acid solution

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·         10 gm of granulated zinc is added (arsenic free).

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·         Keep the solution aside for 40 minutes.

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·         Stain obtained on mercuric chloride paper.

·         A known quantity of dil. Arsenic solution in water and stannated HCl is kept in wide mouthed bottle.

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·         Add 1 gm of KI.

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·         5 ml of stannous chloride acid solution

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·         10 gm of granulated zinc is added (arsenic free).

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·         Keep the solution aside for 40 minutes.

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·         Stain obtained on mercuric chloride paper.

Observation

·         If the stain produce by the test is no deeper than the standard stain, then the sample complies limit test for arsenic.

LIMIT TEST FOR HEAVY METALS

·         The quantity of heavy metals contained as impurities in drugs.

·         The heavy metals are the metallic inclusions that are darkened with sodium sulphate in acidic solution or hydrogen sulfide saturated solution, as their quantity is expressed in terms of the quantity of lead (pb).

Methods

A.      For colour substances

B.      For colorless substances

C.      For substances which form colored and colorless with NaOH

D.      For remaining substances

Apparatus

·         Nessler cylinder

·         Glass rod

·         Stand

Chemicals required

·         Dilute acetic acid (CH3COOH)

·         Dilute ammonia (10% v/v)

·         Hydrogen sulfide solution

·         Standard lead solution

·         Lead nitrate stock solution

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 IP is 20 PPM.

Procedure

·         To take two 50 ml of Nessler cylinders.

·         Label one as TEST and other as STANDARD.

Method-A

Principle

 

·         It is based on the reaction between heavy metals and hydrogen sulfide (H2S) in acidic medium to produce metal sulfide

·         pbs produce brown colour.

·         pH should be determined at 3-4 with acid or dil. Ammonia.

Procedure

Test

Standard

·         place 25 ml of solution prepared for the test.

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·         Adjust pH between 3-4 with dilute CH3COOH OR dilute ammonia solution and dilute with water to produce 35 ml and mix.

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·         Add 10 ml of freshly prepared hydrogen, mix and dilute to 50 ml with water.

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·         Allow to stand for 5 minutes.

·         Pipette 1.0 ml of lead standard solution (20 ppm pb) and dilute with water to produce 25 ml.

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·         Adjust pH between 3-4 with dilute CH3COOH OR dilute ammonia solution and dilute with water to produce 35 ml and mix.

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·         Add 10 ml of freshly prepared hydrogen, mix and dilute to 50 ml with water.

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·         Allow to stand for 5 minutes.

 Observation

·         The colour produced with the test solution is not more intense than that produce with the standard solution.

Method-B

Principle

·         This method is also depending upon reaction between heavy metals ad hydrogen sulfide in acidic medium to produce metallic sulfide and hydrogen gas.

·         pbs produce brown colour.

·         pH should be determined at 3-4 with acid or dil. Ammonia.

Procedure

Test

Standard

·         weight in a suitable crucible, the quantity of the substance specified in the monograph, add sufficient H2SO4 to wet the sample. Ignite carefully at low temperature until thoroughly charred.

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·         after charred add 2 ml of nitric acid and 5 drops of H2SO4 and heat continuously until white fumes are no longer evolved.

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·         ignite preferably in a muffle furnace at 500-6000C until the carbon is completely burnt off.

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·         cool, and add 4 ml of HCl, cover and digest on a water bath for 15 minutes. Uncover and evaporate to dryness.

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·         moisten the residue with 1 drop of HCl and 10 ml of hot water for 2 minutes.

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·         Add ammonia solution drop wise until the solution is just alkaline to litmus paper and dilute to 25 ml with water and adjust the pH at 3-4 with dilute acetic acid, and filter it.

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·         Add filtrate and washing in 50 ml Nessler cylinder and dilute to 35 ml with water and mix.

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·         Add 10 ml of freshly prepared H2S solution, mix and allowed to stand for 5 minutes.

·         Pipette 1.0 ml of lead standard solution (20 ppm pb) and dilute with water to produce 25 ml.

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·         Adjust pH between 3-4 with dilute CH3COOH OR dilute ammonia solution and dilute with water to produce 35 ml and mix.

↓

·         Add 10 ml of freshly prepared hydrogen, mix and dilute to 50 ml with water.

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·         Allow to stand for 5 minutes.

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·         Adjust pH with dilute acetic acid OR dilute ammonia solution to 3-4 and dilute to 35 ml with water.

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·         Add 10 ml of freshly prepared hydrogen sulfide solution, mix and dilute to 50 ml with water and allow to stand for 5 minutes.

 Observation

·         The colour produced in the test solution is not more intense than standard.

Method-C

Principle

·         This method is based upon reaction between sodium sulfide and heavy metal to form metal sulfide in alkaline medium.

·         Here lead sulfate produces brown colour.

Procedure

Test

Standard

·         Place 25 ml of the solution prepared for the test as directed in monograph. OR dissolve the specified quantity of the substance under the examination in a mixture of 20 ml of H2O and 5 ml of dilute NaOH and dilute to 50 ml with H2O

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·         Add 5 drops of sodium sulfide and mix it.

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·         Allow to stand for 5 minutes.

·         Pipette 1.0 ml of lead standard solution (20 ppm pb.) and 5 ml of dilute NaOH. Dilute with H2O to produce 50 ml.

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·         Add 5 drops of sodium sulfide and mix it.

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·         Allow to stand for 5 minutes.

 Observation

·         The colour produce with the test solution is not more intense than that produce with the standard solution.

Method-D

Principle

·         This method is based on the principle on the reaction between thioacetamide and heavy metal in acidic medium.

Procedure

Test

Standard

·         Prepare a solution as directed in the individual monograph and pipette 12 ml into a Nessler cylinder.

↓

·         Add 2 ml of acetate buffer pH 3-5 and mix.

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·         Add 1.2 ml of thioacetamide reagent and allow to stand for 2 minutes. And observed it.

·         Pipette 10.0 ml of either lead standard solution (1 ppm p.) OR lead standard solution (2 ppm pb.) into Nessler cylinder.

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·         Add 2 ml of acetate buffer pH 3-5 and mix.

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·         Add 1.2 ml of thioacetamide reagent and allow to stand for 2 minutes. And observed it.

 Observation

·         The colour produced in the test solution is not more intense than that produce with the standard solution.

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