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Limit test...........................pptx
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- 2. Limit tests: Testsbeing used to identify the impurity. Tests being used to control the impurity. Definition: Limit tests are quantitative or semi quantitative test designed to identify and control small quantities of impurities which are likely to be present in the substances.
- 3. Factors affecting limittests: Specificity of the tests Sensitivity Control of personal errors (Analyst errors) Types: Tests in which there is no visible reaction Comparison methods Quantitative determinations
- 4. Turbidity means cloudinessor haziness.
- 5. Limit test forIRON: Limit test of Iron is based on the reaction of iron in ammonical solution with thioglycolic acid in the presence of citric acid to form iron thioglycolate (Ferrous thioglycolate complex) which produces pale pink to deep reddish purple color in alkaline media. Thioglycolic acid is used as a reducing agent.
- 6. The color ofthe Ferrous thioglycolate complex fades in the presence of air due to oxidation. Also, the color is destroyed in presence of oxidizing agents and strong alkalis. The purple color is developed only in alkaline media. 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.
- 7. Test sample Standardcompound 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 Procedure: Note: All the reagents used in the limit test for Iron should themselves be iron free.
- 8. Observation: • The purplecolor 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 forms complex with metal cation and helps precipitation of iron by ammonia 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 color is visible only in the alkaline media. • The color is not visible in acidic media as ferrous thioglycolate complex decomposes in high acidic media.
- 9. Limit test forCHLORIDE: • The test is used to limit the amount of Chloride as an impurity in inorganic substances. 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.
- 10. The silver chlorideproduced in the presence of dilute Nitric acid makes the test solution turbid. The extent of turbidity depending upon the amount of Chloride present in the substance is compared with the standard opalescence produced by the addition of Silver nitrate to a standard solution having a known amount of chloride and the same volume of dilute nitric acid as used in the test solution.
- 12. Test sample Standardcompound • 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
- 13. Observation: • The opalescenceproduce 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 as dilute HNO3 is insoluble in AgCl.
- 14. • Pharmacopoeia doesnot prescribe any numerical value of limit test for chlorides, sulphate and iron because limit test is based on the simple comparison of opalescence or colour between the test and standard solution prescribed according to pharmacopoeia. • In this type of limit test, the extent of turbidity or opalescence or colour produced in influenced by the presence of other impurities present in the substance and also by variation in time and method of performance of test. • Thus the pharmacopoeia does not prescribe any numerical value of the limit test.
- 15. • The limittest involve simple comparisons of opalescence, turbidity, or colour with standard. • These are semi-qualitative reactions in which extent of impurities present can be estimated by comparing visible reaction response of the test and standard. • By this way, extent of reaction is readily determined by direct comparison of test solution with standard. So pharmacopoeia prefers comparison methods.
- 16. Limit test forsulphate: • The Sulfate Limit Test is designed to determine the allowable limit of sulfate contained in a sample. 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. SO4 -2 + BaCl2 --------------------------- BaSO4 + 2Cl-
- 17. • Then comparisonof turbidity is done with a standard turbidity obtained from a known amount of Sulphate and same volume of dilute Hydrochloric acid have been added to both solutions. • The barium chloride 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. Potassium sulphate has been added to increase the sensitivity of the test.
- 18. Test sample Standardcompound 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 Procedure:
- 19. • Observation: • Theturbidity 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 and so produces a more uniform opalescence.
- 20. Limit test forArsenic: • Arsenic is a well known undesirable and harmful impurity which is present in medicinal substances. • All pharmacopoeias prescribe a limit test for it. • Pharmacopoeial method is based on the Gutzeit test. • All the special reagents used in the limit test for Arsenic are marked and distinguished by letter ‘As T’, which means that they all should be Arsenic free and should themselves conform to the test for Arsenic
- 21. Principle: • Limit testof 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 (H3AsO4) in the sample is reduced to arsenious acid (H3AsO3) by reducing agents like potassium iodide, stannous acid, zinc, hydrochloric acid, etc. Arsenious acid is further reduced to arsine (gas) (AsH3) by hydrogen and reacts with mercuric chloride paper to give a yellow stain. Substance + dil HCl ------------------------- H3AsO4 (contains Arsenic impurity) 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.
- 22. • When thesample is dissolved in acid, the Arsenic present in the sample gets converted to Arsenic acid. • By action of reducing agents like Potassium iodide, stannous acid etc., Arsenic acid gets reduced to arsenious acid. • The nascent hydrogen formed during the reaction, further reduces Arsenious acid to Arsine gas, which reacts with mercuric chloride paper, giving a yellow stain.
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- 24. Apparatus: It ishaving 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 cm long. External diameter=0.8 cm Internal diameter=0.65 cm The tube is constricted at its lower end extremity to about 1 mm diameter and there is blown a hole, not less than 2 mm in diameter, in the side of the tube near the constricted part. The upper end of the glass tube is fitted with two rubber bungs(25 mm x 25 mm), 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 the second bung has to be fitted upon the first bung in such a way that the mercuric chloride paper gets exactly sandwiched between the central perforation of the two. The bungs are kept in close contact by using rubber band or spring clip 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. An important feature has been the standardization of the area of Mercuric chloride paper which is exposed to the reaction of arsine gas.
- 25. Test sample Standardcompound The test solution is prepared by dissolving specific amount in water and stannated HCl (arsenic free) and kept in a wide mouthed bottle. A known quantity of dilute arsenic solution in water and stannated HCl (arsenic free) is kept in wide mouthed bottle. 1 g of KI 1 g of KI 5 ml of stannous chloride acid solution 5 ml of stannous chloride acid solution 10 g of granulated zinc is added (all this reagents must be arsenic free). Keep the solution aside for 40 min 10 g of zinc is added (all this reagents must be arsenic free). Keep the solution aside for 40 min
- 26. • Stain obtainedon mercuric chloride paper is compared with standard solution. Standard stain must be freshly prepared as it fades on keeping. • Inference: If the stain produced by the test is not deeper than the standard stain, then sample complies with the limit test for Arsenic. Reasons: • Stannous chloride is used for complete evolution of arsine. • Zinc, potassium iodide and stannous chloride is used as a reducing agent. • Hydrochloride 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.
- 27. Use ofstannated Hydrochloric acid: • If pure zinc and HCl are used, the steady evolution of gas does not occur. • This produces improper stain (e.g slow evolution produces short but intense stain while rapid evolution of gas produces long but diffused stain.) So, to get steady evolution of gas, stannated hydrochloric acid is used. Use of Lead Acetate solution: • H2S gas may be formed during the experiment as zinc contains sulphides as impurities. • It gives black stain to HgCl2 paper and so will interfere the test. Hence, gases evolved are passed through cotton wool plug moistened with lead acetate, where H2S gas is trapped as PbS. Use of Potassium iodide: • KI is converted to HI which brings about reduction of unreacted pentavalent arsenic to trivalent Arsenic. Thus, reproducible results can be obtained. If it is not used then some pentavalent Arsenic may remain unreacted.
- 28. Limit test forheavy metals • 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 should not exceed the heavy metal limits given under the individual monograph. • 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 term of ppm of lead i.e. the parts of lead per million parts of the substance being examined.
- 29. • In substancesthe proportion of any such impurity (Heavy metals) has been expressed as the quantity of lead required to produce a color of equal depth as in a standard comparison solution having a definite quantity of lead nitrate. • The quantity is stated as the heavy metal limit and is expressed as parts of lead (by weight) per million parts of the test substance. • The limit test for heavy metals has been based upon the reaction of the metal ion with hydrogen sulphide, under the prescribed conditions of the test causing the formation of metal sulphides. • These remain distributed in colloidal state, and give rise to a brownish coloration.
- 30. • I.P limitfor heavy metals in 20 ppm. • The test solution is compared with a standard prepared using a lead solution (as the heavy metal).The metallic impurities in substance are expressed as parts of lead per million parts of substance. IP has adopted 3 methods for this: • Method I: The method is applicable for the samples which give clear colourless solutions under specified conditions of test. • Method II: The method is applicable for the samples which DO NOT give clear colourless solutions under specified conditions of test. • Method III: Used for substances which give clear colourless solutions in sodium hydroxide medium.
- 31. Limit test forlead: • 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 diphenyl thiocarbazone (dithizone) in alkaline solution to form lead dithizone complex which is red in color.
- 32. • Dithizone inchloroform, is able to extract lead from alkaline aqueous solutions as a lead dithizone complex (Red in colour) • The original dithizone is having a green colour in chloroform while the lead-dithizone is having a violet color. So, resulting color at the end of the process is read. • The intensity of the color of complex is dependant upon the amount of lead in the solution. • The color of the lead-dithizone complex in chloroform has been compared with a standard volume of lead solution, treated in the same manner. • In this method, the lead present as an impurity in the substances, gets separated by extracting an alkaline solution with a dithizone extraction solution. • The interference and influence of the other metal ions has been eliminated by adjusting the optimum pH for the extraction by employing Ammonium citrate/ potassium cyanide.
- 33. A known quantityof 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 Test sample Standard compound
- 34. Observation: • The intensityof the color of complex, is depends on the amount of lead in the solution. • The color produced 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 by extracting an alkaline solution with a dithizone extraction solution.
- 35. Aq. Ammonia isadded in limit test of lead: • Pb+ S-------------PbS • In limit test of lead, PbS is produced by addition of standard H2S , to the solution containing lead. • pH 3-4 is necessary for the precipitation of PbS. So aq. Ammonia / acetic acid is added to maintain that pH.
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