Limit Test - Pharmaceutical Analysis 1 B. Pharma 1st semester

Limit Test

Objective

At the end of this lecture, student will be able to

• Define the term Limit test

• Explain principle and procedure involved in the limit test chlorides, sulphates, iron, heavy metals, Arsenic, lead

• Explain the modified procedures for limit test for chlorides and sulphates

Limit Test

The quantitative or semi-quantitative tests designed to identify and control small quantities of impurity which are likely to be present in the substance

Importance of limit test

• Detect even small quantities of impurities

• Specific to impurities

• Simple test involves visible reactions

• Sensitivity of the limit test favours reproducible results

• Direct comparison with standard

Types of limit test

1. Test in which no visible reaction

2. Comparison method

3. Quantitative determinations

Comparison Method

• Standard containing a definite amount of impurity

• Set up at the same time

• Under same conditions as the test

• Direct comparison

E.g. Limit test for chlorides and sulphates etc

Important Limit Tests

 Limit test for Chlorides

 Limit test for Sulphates

 Limit test for Iron

 Limit test for Heavy metals

 Limit test for Arsenic

Limit test for Chlorides

Source: Tap water, chemicals like hydrochloric acid during manufacturing process.

Principle:

Sample –

dil. HNO ₃

Cl^- + AgNO₃ --------------------à AgCl + NO ₃ ^-

                                                                                   Silver chloride(opalescence)

Standard-

 dil. HNO ₃

NaCl + AgNO₃ --------------------à AgCl + NO ₃ ^-

Sample	Standard
Chlorides + silver nitrate â	sodium Chloride  + silver nitrate â
Conc.HNO3 â	Conc.HNO3 â
opalescence of silver chloride	opalescence of silver chloride

 

Compare (against dark back ground)

Opalescence of the sample solution is less than the standard solution à Sample passes the limit test                                                                                  

Opalescence of sample solution is more than the standard solution à Sample fails the limit test

Procedure for Limit Test for Chloride

Standard	Sample
•10 ml of standard sodium chloride solution.	•10 ml of sample solution
•10 ml of dil HNO3	•10 ml of dil HNO3
•Dilute with distilled water.	•Dilute with distilled water.
•Stir	•Stir
•1 ml of 0.1 M AgNO3 solution.	•1 ml of 0.1 M AgNO3 solution.
•Mix thoroughly	•Mix thoroughly
• allow to stand for 5 minutes.	• allow to stand for 5 minutes

Limit Test for Sulphates

• Source: Tap water, chemicals like sulphuric acid during manufacturing process

• Principle:

Barium sulphate reagent

•   Composition of Barium sulphate reagent

Barium chloride - Used as reagent to convert sulphate to barium sulphate

Potassium sulphate - increases the sensitivity of the test opalescence

Alcohol - prevents super saturation and produces uniform

Sample –

dil.CH3COOH

SO₄^2- + BaCl₂ ---------------------àBaSO₄ + 2Cl^-

                                                                                          barium sulphate (opalescence)

Standard –

dil.CH3COOH

K₂SO₄ + BaCl₂ ---------------------àBaSO₄ + 2Cl^-

25 ppm

Compare (against a dark background)

Opalescence of the sample solution is less than the standard solution à sample passed

Opalescence of the sample solution is more than the standard solution à sample failed

Procedure for Limit Test for Sulphates

Standard	Sample
• 1 ml of 25% w/v barium chloride	• 1 ml of 25% w/v barium chloride
•1.5 ml of ethanolic potassium sulphate	•1.5 ml of ethanolic potassium sulphate
•15 ml of standard sulphate	• 15 ml of sample sulphate
•one drop /0.15 ml of 5M acetic acid	•one drop /0.15 ml of 5M acetic acid
•Stir well	•Stir well
• dilute with distilled water.	• dilute  with distilled water
•Mix well	•Mix well
•allow to stand for 5 minutes	•allow to stand for 5 minutes

Limit Test for Iron

•   Source: manufacturing process, vessels

Fe₂⁺ + Thioglycollic acid

               Ammonia         â          iron free citric acid

Ferrous thioglycollate (reddish purple colour)

â

Comparison of colour intensity [sample and standard (ferric ammonium sulphate, 20ppm)]

Colour intensity of the sample solution is less than the standard solution à Sample passes the limit test                         

Colour intensity of the sample solution is more than the standard solution à Sample fails the limit test

Procedure for Limit  Test for Iron

Standard	sample
•2ml of standard iron solution	•2ml of sample iron solution
•2 ml of 20%w/v iron free citric acid.	•2 ml of 20%w/v iron free citric acid.
•one drop /0.1 ml of thioglycollic acid.	•one drop /0.1 ml of thioglycollic acid.
•Stir	•Stir
•Make the solution alkaline	•Make the solution alkaline
•by adding ammonia drop wise	•by adding ammonia drop wise
•until red litmus turns to blue.	•until red litmus turns to blue.
•Make up with distilled water.	•Make up with distilled water.
•Stir.	•Stir.

  

Limit test for Heavy metals

Sources

• Deliberately added as catalysts or reagents

• Natural occurrence in source materials

• Processing equipment

Methods

Substances which give clear and colourless solution Reagent – H2S

Substances which do not give clear and colourless solution Reagent - H2S

Substances which give clear and colourless solution Reagent – Na2S

Substances in which sample itself reacts with hydrogen sulphide reagent

Reagent– Thioacetamide

Limit test for Heavy Metals

P^H = 3 ^- 4

2M ⁿ⁺ + nH₂S ------------------------------à M₂Sn + 2 nH⁺

dil.CH₃COOH and NH₃

P^H = 3 ^- 4

Pb(NO₃)₂ + H2S  ------------------------------à PbS + 2HNO₃

dil.CH₃COOH and NH₃

Heavy metals + Hydrogen sulphide à Respective sulphides, brown to light brown colour à

1. Colour intensity of the sample solution is less than the standard solution à Sample passes the limit test                      

2. Colour intensity of the sample solution is more than the standard solution à Sample fails the limit test

Procedure for Limit test for Heavy Metals

Standard	Sample
•1 ml of standard lead nitrate solution	•sample solution
•Dilute up to 25 ml with distilled water.	•Dilute up to 25 ml with distilled water.
•Adjust the pH between 3-4 using ammonia and dilute acetic acid.	•Adjust the pH between 3-4 using ammonia and dilute acetic acid.
• Dilute with water to about 35 ml	• Dilute with water to about 35 ml
• Mix	• Mix
•10 ml of H2S	•10 ml of H2S
•Mix	•Mix
•Dilute with distilled water	•Dilute with distilled water
• Allow stand for 5 minutes.	• Allow stand for 5 minutes.

 

Limit test for Arsenic

• Source: Natural occurrence in source materials or processing equipments or solvent

As 3+ or As 5+ --------à   arsine gas --------à  mercuric chloride --------à stains paper yellow

Standard [arsenic trioxide (1 ppm)]                                                                            

• compare sample stain and standard stain (arsenic trioxide, 1ppm)

Colour intensity of the sample stain is less than the standard stain colour intensity

 Colour intensity of the sample solution is more than the standard stain colour intensity

Chemical Reaction Involved in Limit test for Arsenic

• As 3+ ----------à  H3 AsO3 (arsenious acid)

• As 5+ ----------à H3 AsO4 (arsenic acid)

• H3 AsO4 ----------à H3 AsO3

• Zn +2 HCl ----------à ZnCl2 + 2[H]

                              Reduction

• H3AsO3 +6[H] ----------à AsH3 + 3H2O

• 2AsH3 + HgCl2 ----------à Hg (As H2)2    + 2HCl

Apparatus Used for Limit Test for Arsenic

Procedure Involves in Limit Test for Arsenic

Standard	Sample
1 ml of standard arsenic solution in 50 ml of distilled water.	Dissolve 1 g of NaCl in 50 ml of distilled water
10 ml of stannated HCl solution.	10 ml of stannated HCl solution.
1g of potassium iodide.	1g of potassium iodide.
1 g of zinc granules or granulated zinc. Stopper the container with a capillary tube, fitted with lead acetate cotton wool and rubber bungs, fitted with mercuric chloride paper.	1 g of zinc granules or granulated zinc. Stopper the container with a capillary tube, fitted with lead acetate cotton wool and rubber bungs, fitted with mercuric chloride paper.
Allow the container to stand at 40ºC for 40 minutes.	Allow the container to stand at 40ºC for 40 minutes.

 

Compare the sample stain with standard stain and report

Precautions for Performing Limit Test

• Zinc granules are used

• The reaction should be carried out at 40°C for 45 minutes

• Placing a lead acetate soaked cotton plug

H2S + HgCl 2 --------à HgS      +   2HCl

Mercuric sulphide brown stain interferes the test

H2S   + Pb(CH3COOH) 2 --------à PbS     +  2CH3COOH

• H2S is trapped by lead acetate  

Limit Test for Lead

Source: Natural occurrence in source materials

              Processing equipments

              Solvent or process (lead chamber process)

Extract lead from an alkaline aqueous solution

                                 â dithizone (green)

Lead dithizonate(red colour)

â

Green colour of unreacted dithizone + red colour of complex

â

Violet colour shade

Comparison with standard solution prepared (lead nitrate solution, 1ppm)

Colour intensity of the sample solution is less than the standard solution colour intensity àSample passes the limit test            

 Colour intensity of the sample solution is more than the standard solution colour intensity àSample fails the limit test

Procedure Involved in Limit teat for Lead

Transfer the required volume of the prepared sample to a separating funnel.

â

Add 6 ml of ammonium citrate Sp and 2 ml of hydroxylamine hydrochloride

â

Make the solution just alkaline by addition of strong ammonia

â

Add 2 ml of KCN solution

â

Extract with several quantity each of 5 ml of dithizone extraction solution

â

Until dithizone extraction solution retains its green colour

â

Shake the combined dithizone solution with 30 ml of 1% v/v nitric acid

â

Discard chloroform layer

â

Add 5 ml of dithizone standard (lead) solution and shake for 30 seconds

â

The colour of chloroform layer is not more intense than that obtained by treating in the same manner a volume of lead nitrate solution, 1 ppm

 

 

Modified procedure for limit test for chlorides and sulphates

• Deeply coloured substances

• Alkaline substance

• Salts of organic acids

Potassium permanganate

Deeply coloured substance is decolourised

â

Reacting with alcohol (reduction process)

â

Solution is filtered

â

The colourless filtrate is collected

â

The normal chloride limit test is carried out

Chemical Reaction

2KMnO4 + 3CH3CH2OH -----------à 2MnO2 + 3CH3CHO + 2H2O

Sodium Bicarbonate

•     Bicarbonate is first dissolved in suitable solvents

•     Treated with conc. HNO3

•     The effervescence ceases

• The normal chloride limit test is carried out

NaHCO3 + HNO3 ---------------à CO2 + 2NaCl + H2O

Sodium Benzoate

Salts of organic acids

â

Treated with conc. HNO3

â

Converted to their respective acids

â

Filtrate is employed in the limit test for chlorides

 

Chemical Reactions

• Sulphates- Conc. HCl is used

NaHCO3 + HCl ------------à CO2 + 2NaCl + H2O

Summary

• Limit test is the quantitative or semi-quantitative tests to identify & control small quantities of impurity

• Importance of limit tests

Detect even small quantities of impurities, Specific to impurities, Simple test involves visible reactions, Direct comparison with standard

• Limit test for Chlorides

Chlorides + silver nitrate in presence of nitric acid forms silver chloride forms opalescence

• Limit test for Sulphates

Sulphates + barium chloride gives barium sulphate

• Limit test for Iron

Ferrous ion + thioglycollic acid in presence of ammonia gives ferrous thioglycollate

• Colour intensity is compared with the standard

• If opalescence/colour intensity   of test solution is more than the standard sample fails the limit test

• Limit test for Heavy metals

Method A

M + H2S gives metallic sulphide and Colour intensity is compared

Standard used is lead nitrate

Colour intensity of test is less than standard then sample passes the limit test

• Limit test for arsenic

Arsenic is converted into arsine gas + mercuric chloride paper stains yellow mercuric arsenite

Colour intensity is compared with arsenic trioxide

Colour intensity is less than the standard sample passes the limit test

Lead acetate cotton plug is to trap sulphur impurity (H2S) as lead sulphide

• Limit test for Lead

Lead + Dithizone gives lead dithizonate(red colour)

Green colour of unreacted dithizone + red colour of complex gives violet colour

Colour intensity is compared with standard (lead nitrate), if it is more than sample fails the limit test

• Modification tests for chlorides and sulphates

• Deeply coloured substance like KMnO4 should be decolourised with alcohol and filtrate is used for normal limit test

•Alkaline substances should be treated with conc. HNO3/conc. HCl till effervescence ceases and use it for normal limit test

• Salt of organic acids should be treated with conc. HNO3/conc. HCl and filtrate is used for normal limit test