UNIT I_History of pharmacopoeia and limit test

As per the SPPU Syllabus: For Pharmaceutical Inorganic Chemistry

1. Impurities in Pharmaceutical Substances By… Prof. Sonali R. Pawar Assistant Professor, Pharmaceutical Chemistry Department , Pharmaceutical Inorganic Chemistry 1 UNIT I

2. Content: History of Pharmacopoeia Sources and types of impurities Principle involved in the limit test for Chloride Sulphate Iron Arsenic lead and Heavy metals Modified limit test for chloride and sulphate IMPURITIES IN PHARMACEUTICAL SUBSTANCES 2

3. Pharmaceutical chemistry deals with the structure, chemical nature, composition, preparation, studies of physical and chemical properties, methods of quality control and conditions of their uses. The subject is further sub-divided into various branches like: Inorganic chemistry . Biochemistry Organic chemistry Analytical chemistry Physical chemistry Phytochemistry Medicinal chemistry 3

4. Now a days compounds of various metals like iron, arsenic, lead, mercury & copper were used for medicinal purpose. Hence, a sound knowledge of chemistry is required for understanding and following the recent developments in medicine and pharmacy. Pharmaceutical chemistry thus plays an important role in deciding the physiochemical properties, conditions for the storage and dispensing of drugs. 4

5. PHARMACOPOEIA Pharmacopoeia: the word derives from the ancient Greek “ pharmako poi ia ” from pharmako - ″drug″, followed by the verb-stem poi - ″make″ and finally the abstract noun ending - ia . These three elements together can be rendered as ″ drug- mak - ing ″ or ″ to make a drug ″. A pharmacopoeia , pharmacopeia , or pharmacopoea is a legally binding collection (legal document, monograph, official publication/book), prepared by a national or regional authority, of standards and quality specifications for medicines used in that country or region (e.g., IP, BP, USP). quality specification: such as identity strength or amount and purity of medicines 5

6. Pharmacopoeia is the official book of standards for drugs prepared by any country or regulatory body to specify the standards of identity, purity and strength for the drugs imported, manufactured or distributed throughout the country or a specific region. It is a book containing collection of monographs and published by an authorized body like government or Pharmaceutical society. T h e ter m Ph armac op o e ia c o m e s f r o m t he Greek w o r d “Ph a r mak o n ” m e a n i n g dr ug a n d “ Po i e o ” m ea ni n g make, a n d t h e c o m b i n a t i o n m ean s a n y formula or standards required to make a drug. PHARMACOPOEIA 6

7. Organization, region or country Name of pharmacopoeia World Health Organization The International Pharmacopoeia (Ph. Int.) India Indian Pharmacopoeia (IP) European Union European Pharmacopoeia (Ph. Eur.) United Kingdom (UK) British Pharmacopoeia (BP) China Pharmacopoeia of the People's Republic of China Japan The Japanese Pharmacopoeia (JP) United States of America United States Pharmacopeia (USP) In addition to these some other documents prescribing standards are: British Pharmaceutical Codex (B.P.C.) & National Formulary in America (U.S.N.F.) 7

8. • A monograph is a collection of detailed information on a particular drug, its dosage forms and methods of analysis. • Monographs provide the following information: Main title/synonyms Chemical formula/formula weight Therapeutic category Dosage Description Solubility Minimum standards of purity Identification tests Tests for purity/limit tests for impurities Methods of assay Storage condition Packaging and labeling 8

9. Importance of pharmacopoeia 9 To maintain the uniformity and control the standards of the drugs available in market. Avoid adulterated drugs. Complete information on drugs and their dosage forms. Reference for laboratory, industry and academic institutions.

10. Formulary 10 • F o rm u l a r i es a r e t h e l i s t of d r u g s or co l le ct i o n s of formulas for the compounding of medicinal • • preparations. F o rm u l a r i es c o n tai n s m o r e c o m p r ehe n s iv e d e tails on therapeutics. Collectively these books are known as drug compendia. Pharmacopoeias + Formularies = Drug Compendia

11. Drug compendia 1. Official compendia Official compendia are the compilation of drugs and other substances recognized as legal standards of purity, quality and strength by government agency of respective countries. British Pharmacopoeia (BP) British Pharmaceutical Codex (BPC) United States Pharmacopoeia (USP) Indian Pharmacopoeia (IP) Japanese pharmacopoeia (JP) 11

12. 2. Non-official compendia The books other than official drug compendia which are used as secondary reference sources for drugs and other related substances are known as non-official drug compendia. Merck Index Martindale (The extra Pharmacopoeia) 12

13. PHARMACOPOEIA The term Pharmacopoeia first appears as a distinct title in a work published in Basel, Switzerland in 1561 by Dr A. Foes. But does not appear to have come into general use until the beginning of the 17th century. Today’s pharmacopoeias focus mainly on assurance of quality of products by various tools of analytical sciences. Drugs & Cosmetic Act 1949 and Rules 1945. IP is published by the Indian Pharmacopoeia Commission, the Standards Setting Institution for Drugs. It is an autonomous body under the Ministry of Health & Family Welfare. To keep pharmacopoeia up-to-date, pharmacopoeial commissions constantly update pharmacopoeia Once in every four years . 13

14. Indian Pharmacopoeia (IP) 14 Indian Pharmacopoeia is the official book of standards for drugs to define identity, purity and strength for the drugs imported, manufactured for sale, stocked or distributed in India. In 1946 Government of India Published Indian Pharmacopoeial List which served as supplement to BP. Indian Pharmacopoeia was prepared by Indian Pharmacopoeia Committee formed in 1948.

15. Indian P h ar m ac o p o e i a 15 Year of P ub l ica t i on 1 st Edition 1955 986 monographs Supplement to 1 st Edition 1960 2 nd Edition 1966 890 monographs Supplement to 2 nd Edition 1975 3 rd Edition 1985 Consists of 2 Volumes IUPAC system of nomenclature Analytical Techniques were included e.g: Electrophoresis, Fluorimetry Instrumental Analysis were included e.g: UV spectroscopy Dissolution of 6 tablets included Limit tests for microbial contamination Addendum to 3 rd Edition 1989 Addendum to 3 rd Edition 1991 Co n te n ts

16. Indian Pharmacopoeia Year of P ub l i c ati on Contents 4 th Edition 1996 1. Consists of 2 Volumes 2. Included 1149 monographs 3. Included new monographs e.g: cream, gels, eye drops 4. Included method of preparation and analytical methods like HPLC 5. Include In Process Quality control Addendum to 4 th Edition 2000 Supplement to 4 th Edition 2000 Veterinary Products Addendum to 4 th Edition 2002 Addendum to 4 th Edition 2005 5 th Edition 2007 Consists of 3 Volumes Addendum to 5 th Edition 2008 6 th Edition 2010 Consists of 3 Volumes Addendum to 6 th Edition 2012 7 th Edition 2014 Consists of 4 Volumes with DVD Addendum to 7 th Edition 2015 Addendum to 7 th Edition 2016 16

17. Indian National Formulary It is a reliable reference book on drugs formulations for the practicing physicians/clinicians, pharmacists, clinical pharmacists, nurses and others engaged in healthcare profession. Indian Pharmaceutical Codex 1953 It is a book containing detailed information on indigenous drugs of India. 17

18. British Pharmacopoeia (BP) British Pharmacopoeia is the source of official standards of drugs in UK and other parts of the world. Commission . Since then Pharmacopoeial commission is reconstituted from time to time and new editions of British Pharmacopoeia are published. 18 It was first published by G e n er al M e d ic in e C o un c i l and was later done b y P h a r mace u t ical

19. British P h ar m ac o p o e i a 19 Year of Pu b l i ca tion C o n te n ts First Publication 1864 extracts, crude drugs, galenicals 14 th Edition 1988 2100 monographs Contains 2 Volumes Volume I: monographs of medicinal and pharmaceutical substances Volume II: formulations, blood products, appendices It is now published annually and consists of 6 volumes.

20. British Pharmaceutical Codex (BPC) 20 British Pharmaceutical Codex was prepared as a reference book to physicians and dispensing pharmacists in 1907 as per the Council of Pharmaceutical Society. Since then subsequent revisions of these books are published. The decision of medicine commission stated that there should be only one book of standards of medicine, so BPC was discontinued. Later BPC was published as “The Pharmaceutical Codex” and plans to be encyclopedia.

21. The BPC differs from BP in: 21 BPC contains more drugs and preparations. It contains additional information on standard of drugs, surgical dressing, pharmaceutical preparations, etc It provides action and uses of drugs. It contains formula and preparation methods of some other formulations.

22. British National Formulary (BNF) 22 British National Formulary is a source of essential information on drugs and medicines published by pharmaceutical society of Great Britain and British Medical Association. Pharmacological classification of drugs are given. It includes preparations as per Pharmaceutical forms. It provides information about actions, uses, dosage & adverse reactions.

23. United States Pharmacopoeia (USP) 23 • The United States Pharmacopoeia and the National Formulary (USP-NF) are recognized as the official compendia and are used as reference books for determining the strength, quality, purity, packaging and labeling of drugs and other related articles. • • Fi r s t U S P was p u b li s h e d i n 182 b y U S Pha rmace u t i cal Convention in English and Latin. It consists of 272 drugs. US P c o nt a in s o v er 340 0 m o n o g raphs f o r d r u g subst ances and products, together with over 160 general chapters that describe • s p ec ifi c p r o ce du res t o s u p p o r t m o n o g raph t e st s and o th er information as well. US P also c o n t ains 1 6 m o n o g raphs and 9 ge n e r a l c hap t e r s o n nutritional supplements.

24. National Formulary (NF) 24 First National Formulary of US was published in 1888 by American Pharmaceutical Association. USP and NF was combined as a single book of drug standards as USP-NF. USP-NF represents 25 th revision of USP & 20 th revision of NF official on 2002. From then USP-NF was published annually. NF covers over 3800 monographs for excipients and dietary supplements.

25. International Pharmacopoeia 25 The International Pharmacopoeia is published by the WHO and is practically used in developing countries. It was prepared to meet the international uniformity and standardization of drugs. International Pharmacopoeia was first published in 1951 in multilanguages ( English, French, German, Japanese, etc.).

26. European Pharmacopoeia (Ph. Eur.) 26 • • The European Pharmacopoeia (Ph. Eur.) is the legal document for the standards of drugs and related substance prepared by the Council of Europe. European Pharmacopoeia Volume I was published in 1969 as first European Pharmacopoeia. It includes more than 2000 specific and general monographs, including various chemical substances , antibiotics , biologicals, v a c c i n e s, herbal, immunosera , radiopharmaceuticals, homeopathic preparations . ,

27. Japanese Pharmacopoeia (JP) 27 Japanese Pharmacopoeia is established and published to regulate the properties and quality of drugs by Ministry of Health, Labour and Welfare of Japan. It consists of general notices, rules for crude drugs, rules for preparations, general tests, processes, apparatus and official monographs. First published in 1886, JP has been revised many times.

28. Introduction Pure Chemical Compound: Having no Foreign matter that is Impurity Chemical Purity means freedom from foreign matter. 28

29. 29 Presence of Impurities in the pharmaceutical substances may produce toxic effects on the body and may also lower down the active strength of the pharmaceutical substance. Impurities commonly in chemical substances include small quantities of lead, Arsenic, Iron, Chloride and sulphate.

30. Sources of Impurities Improper Storage Deliberate Adulteration Atmospheric Contamination Manufacturing Hazards Raw Material Method of Manufacturing 30

31. Types of Impurities 31 Analytically 100 % pure substances are not available and traces of impurities must be present. Normally undesirable foreign materials are present in the pharmaceutical substances.

32. Sources & Types of Impurities Raw material Method of manufacturing (Reaction Vessels, Reagent used, Solvent used, intermediates) Manufacturing hazards (Particulate contamination, process error, packaging error,cross contamination, microbial contamination) Improper storage (Filth, temperature effect, reaction with container) Atmospheric contamination Delibrate adultrance 32

33. TEST FOR PURITY Washing Drying Recrystallization of solid substances from water Sublimation METHODS USED TO PURIFY THE INORGANIC SUBSTANCE Colour, Odour And Test Physico - chemical constants Acidity, alkalinity and PH Moisture content Anions and cations Ash value Loss of ignition Loss on drying 33

34. Impurities: A compound is said to be impure if it is having foreign matter i.e., impurities. Pure chemical compound refer to that compound which is having no foreign matter! Purification of chemicals is an expensive process, substances should not be purified more than required as it brings about waste of time, material and money. In such cases, if it is not possible to eliminate out these impurities completely, attempts have to be made to at-least minimise their concentration! 34 Since the drug and pharmaceutical products concern with health and life of people and animal The prime consideration behind every formulation is that the product should be: Pure as much as possible (absolute purity). Satisfactory clinically. Homogenecity . Safety in its use.

35. Pharmacopoeia prescribe limits for physiologically harmful compound(s)/impurities present in substances/formulations. Impurities commonly found in Medicinal preparations: 1. Activity depressing impurities. e.g., presence of water in hard soap. 2. Due to colouring or flavouring substances , e.g., Sodium Salicylate is discoloured due to phenolic compounds. 3. Humidity – may cause many substances to oxidize. 4. Decrease shelf life. 5. Physical and chemical properties. 6. Impurities due to which substances become incompatible or unfit for medicinal use. (Physical, Chemical & Therapeutically). 7. Toxic impurities . E.g. Lead and arsenic salts. 35

36. SOURCES OF IMPURITIES: Raw, Solvents, Mfg , etc 1. RAW MATERIALS Employed in the Manufacturing of the Pharmaceutical Substance: It is essential to verify the identity of the source (natural e.g. mineral sources, plants, animals, microbes or synthesized from chemicals) of raw materials. In nature minerals rarely occurs in a reasonably pure from. Almost always mixtures of closely related substances occur together Rock salt used for the preparation of sodium chloride is contaminated with small amounts of calcium and magnesium chlorides 36

37. e.g., Barium and Magnesium impurities are found in calcium minerals. Magnesium or Iron compounds are found in zinc. Lead and Heavy metals are found as impurities in many sulphide ores. Chlorides, bismuth salts contains silver copper and lead as impurities. 37

38. SOURCES OF IMPURITIES 2. METHOD OF MANUFACTURE : Reagents used/employed in the manufacturing process: Calcium carbonate contains ‘soluble alkali’ as impurity which arises from the sodium carbonate (Na2CO3) employed in the process of Calcium carbonate. CaCl2 + Na2CO3 → CaCO3 ↓ + 2 NaCl Soluble Soluble Precipitate Soluble Anions like chlorides and sulfates are common impurities in many substances because of the use of tap water , hydrochloric acid and sulphuric acid respectively in processing. Hydrogen peroxide can contaminate the final product with barium ion. (B) Regents used to eliminate other impurities: Barium is used in the preparation of potassium bromide to remove sulphate which in turn - potassium bromide will now be contaminated by traces of barium. 38

39. SOURCES OF IMPURITIES (C) Solvents: Water ! Tap water: Containing impurities of Ca2+, Mg2+, Na+, Cl –, CO3 –2 and SO4–2 in trace amounts. The use of tap water on large scale will lead to the contamination of the final product with these impurities because the impurities will remain in the product even after washings. Demineralized water: is free from above ions. It may have pyrogens , bacterias and organic impurities. So, discourage its use on large scale. Distilled water: It is free from all organic and inorganic impurities and is therefore the best as a solvent but it is quite expensive. Softened water: It is almost free from divalent cations (Ca2+, Mg2+ ) but contains more of Na+ and Cl – ions as impurities. 39

40. SOURCES OF IMPURITIES D) Reaction vessels: The reaction vessels used/employed in the manufacturing process may be metallic such as: copper, iron, cast iron, galvanized iron, silver, aluminium, nickel, zinc, lead, glass and silica. Some solvents and reagents used/employed in the process may react with the metals of reaction vessels, leading to their corrosion and passing traces of metal impurities into the solution & contaminating the final product. Similarly, glass vessels may give traces of alkali to the solvent. 40

41. SOURCES OF IMPURITIES (E) Intermediates: Sometimes, an intermediate substance produced during the manufacturing process may contaminate the final product e.g., Sodium bromide is prepared by reaction of sodium hydroxide and bromine in slight excess. 6NaOH + 3Br2 → NaBrO3 + 5NaBr + 3H2O The sodium bromate an intermediate product is reduced to sodium bromide by heating the residue (obtained by evapourating the solution to dryness) with charcoal. NaBrO3 + 3C → NaBr + 3CO Sodium bromate Sodium bromide If sodium bromate is not completely converted to the sodium bromide then it is likely to be present as an impurity. 41

42. SOURCES OF IMPURITIES (F) Atmospheric contamination during the manufacturing process: Atmosphere may contain: dust of aluminum oxide, sulphur, silica etc. And some gases like carbon dioxide , sulphur dioxide and hydrogen sulphide etc. These may contaminate the final product. e.g., sodium hydroxide readily absorbs atmospheric carbon dioxide when exposed to atmosphere. 2NaOH + CO2 → Na2CO3 + H2O Calcium hydroxide solutions can absorb carbon dioxide from the atmosphere to form calcium carbonate. Ca(OH)2 + CO2 → CaCO3 + H2O 42

43. SOURCES OF IMPURITIES (G) Manufacturing hazards: Contamination from the particulate matter: from sieves, granulating, tabletting and filling machines and the product container. From dirty or improperly maintained equipments. From Cross-contamination of the product. Contamination by microbes: products intended for parenteral administration and ophthalmic preparations are liable to contamination by microbes from the atmosphere sterility testing - provides an adequate control for microbial contaminations in such preparations. Microbial contamination can be controlled by adding suitable antimicrobial and antifungal agents. 43

44. SOURCES OF IMPURITIES Errors in the packaging: Similar looking products, such as tablets of the same size, shape and colour, packed in similar containers can result in mislabeling of either or both of the products. Adequate care should be taken to avoid the handling of such products in the close proximity. Errors in the manufacturing process Cross contamination Microbial Contamination 44

45. 3. Instability of the Product Chemical instability/decomposition: Impurities can also arise during storage when storage conditions are inadequate. This chemical decomposition is often catalyzed by: Light , traces of acid or alkali, traces of metallic Impurities, air oxidation, carbon dioxide and water Vapours . It can easily be predicted from the knowledge of chemical properties of the substance. It can be minimized or avoided by using proper storage procedures & conditions. 45

46. Improper storage: ex. Ferrous sulphate slowly gets changed into insoluble ferric oxide by air and moisture. a)Filth : Contaminated with dust b)Temperature Effects: The rate at which chemical decomposition and physical changes occure in stored products depend upon it. c) Reaction with container material: container material and content . d)chemical instability Atmospheric contamination during the manufacturing process. Deliberate adulteration 46

47. The photosensitive substances should be protected from light by storing them in darkened glass (amber glass bottles) or metal containers thereby inhibiting photochemical decomposition. Materials susceptible to oxidation by air or attack by moisture should be stored in sealed Containers and if necessary the air from the containers can be displaced by an inert gas such as Nitrogen or by adding suitable antioxidants (like in food products ascorbic acid, tocopherol and sodium citrate. 47

48. (B) Changes in physical properties: changes in crystal size and shape, sedimentation, agglomeration and caking of the suspended particles. Particle size and surface area is a critical factor in determining the bioavailability of the low solubility drug. E.g : under dosage and later to over dosage of the drugs (suspensions). & Injectable emulsions on storage may lead to fat embolism. 48

49. (C) Reaction with container material: e.g., salicylic acid ointment must not be packed in metal tubes. alkali-sensitive e.g., atropine sulphate injection must be packed in glass ampoules must not be packed in containers made from soda glass. Plastic containers - plasticizers, particularly in the presence of non-aqueous solvents. Rubber closures are more susceptible to absorb medicaments Temperature: The rate of chemical decomposition and physical changes of stored products depends upon the temperature. 49

50. Types of Errors or Impurities 1. Determinate Errors (Systematic errors): Definite value and underlying (undisclosed/hidden) cause. Can measure & account for these errors. Sources: Impurities in the crude material. Excess of reagent used. Salts from the water used when the preparation is carried out in aqueous solutions. Metallic contamination. Contamination from exposure to the atmosphere. 50

51. Types of Errors or Impurities 2. Indeterminate errors: Result from extending a system of measurements to its maximum. Neither identify the sources of these errors nor predict the magnitudes of individual errors. 51 The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH)

52. Permissible Limits of Impurities Impurities are always present in the Pharmaceutical products. The following points are considered to permit the impurities in these substances: A level of harmful substances is to be determined. A permissible limit, based on the amount to be tolerated of the toxic substances is prescribed. e.g., Heavy metal (Toxic impurities) limits in India 52 P.T.O

53. Heavy metal limits 53 Permissible Limits of Impurities

54. Permissible Limits of Impurities Acceptance criteria for impurities in drug substances : Each identified specified impurity (Determinate errors) Not more than 0.5 per cent Each unidentified impurity (Indeterminate errors) Not more than 0.3 per cent Total impurities Not more than 1.0 per cent 54 Provided it has been determined that the impurities are not toxic. Higher limits may be set if scientifically justified. Acceptance criteria for degradation products in d rug products: Each identified degradation product Not more than 1.0 per cent Each unidentified degradation products Not more than 0.5 per cent Total degradation products Not more than 2.0 per cent Provided it has been determined that the impurities are not toxic. Higher limits may be set if scientifically justified.

55. the limit for harmless impurities are also prescribed depending upon the nature, type and usefulness of the substances. e.g., colouring substances flavouring agents preservatives are the harmless compounds added to the pharmaceutical preparations. 55 Permissible Limits of Impurities Intermediate By-product Transformation product Interaction products Related products Degradation products

56. If there is no emphasis on impurities in kilogram(s) and liters of food taken by an individual in a day 56 Then how come it is a so serious issue for few milligrams of drug(s) consumed in a tiny pill daily?

57. CONTENTS 57

58. LIMIT TEST 58 “Limit test is defined as quantitative or semi quantitative test designed to identify and control small quantity of impurity which is likely to be present in the substance.” Importance of Limit Test To find out the harmful amount of impurities To find out the avoidable/ unavoidable amount of impurities Limit Test Invoves The simple comparison of opalescence, turbidity or colour produced in test with that of fixed slandered. limit test for inorganic compound is carried out so that the amount of inorganic impurity present in the drug/required material do not exceed is prescribed limit test. Limit tests:  Tests being used to identify the impurity.  Tests being used to control the impurity.

59. 59 Factors affecting limit tests : Specificity of the tests Sensitivity Control of personal errors (Analyst errors ) • Test in which there is no visible reaction • Comparison methods • Quantitative determination Types: Tests in which there is no visible reaction Comparison methods Quantitative determinations

60. 60 The limit test 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.

61. LIMIT TEST FOR CHLORIDE 61

62. 62 The test is used to limit the amount of Chloride as an impurity in inorganic substances. LIMIT TEST FOR CHLORIDE 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. Soluble chloride present as impurity The silver chloride produced 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. Cl- + AgNO3 AgCl + NO3 –

63. 63

64. 64 Test sample Standard compound Specific weight of compound is dissolved in water or solution is prepared as directed in the pharmacopoeia and transferred in Nessler cylinder Take 1 ml of 0.05845 % W/V solution of sodium chloride in Nessler cylinder Add 1 ml of nitric acid Add 1 ml of nitric acid Dilute to 50 ml in Nessler cylinder Dilute to 50 ml in Nessler cylinder Add 1 ml of AgNO 3 solution Add 1 ml of AgNO 3 solution Keep aside for 5 min Keep aside for 5 min Observe the Opalescence/Turbidity Observe the Opalescence/Turbidity PROCEDURE

65. 65 Observation The Opalescence Produce 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 HNO 3 is insoluble in AgCl.

66. LIMIT TEST FOR SULPHATE 66

67. 67 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. LIMIT TEST FOR SULPHATE

68. 68 Then comparison of 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.

69. 69 Test sample Standard compound Specific weight of compound is dissolved in water or solution is prepared as directed in the pharmacopoeia and transferred in Nessler cylinder Take 1 ml of 0.1089 % W/V solution of potassium sulphate in Nessler cylinder Add 2 ml of dilute hydrochloric acid Add 2 ml of dilute hydrochloric acid Dilute to 45 ml in Nessler cylinder Dilute to 45 ml in Nessler cylinder Add 5 ml of barium sulphate reagent Add 5 ml of barium sulphate reagent Keep aside for 5 min Keep aside for 5 min Observe the Turbidity Observe the Turbidity PROCEDURE

70. 70 Observation The turbidity 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. Barium sulphate reagent contains barium chloride, sulphate free alcohol and small amount of potassium sulphate.

71. LIMIT TEST FOR IRON 71

72. S.N. Chemical Quantity Apparatus Quantity 1 Ferric Ammonium Sulphate [NH 4 Fe(SO 4 ) 2 ] 0.17 gm Nessler Cylinder 2 2 0.1 N Sulphuric Acid (H 2 SO 4 ) 10 ml Beaker (100 ml) 2 3 Iron Free Citric Acid (C 6 H 8 O 7 ) 2 gm Glass rod 2 4 Thioglycolic Acid (C 2 H 4 O 2 S) 0.5 ml Pipette 2 5 Ammonia Solution (NH 4 OH) 20 ml Stand 1 Standard Solution: Accurately weighted 0.1726 gm of ferric ammonium sulphate dissolved in 10 ml of 0.1 N sulphuric acid (H 2 SO 4 ) and volume make up to 1000 ml with distilled water. Each ml of solution contains 0.02 mg of iron. Citric acid (20%): Dissolve 20 g of iron free citric acid in 100 ml of distilled water. Iron free ammonia solution: Dilute ammonia solution i.e. 10 % ammonia (10 ml dissolve in 100 ml in distilled water). REAGENT PREPARATION 72

73. PRINCIPLE Iron, in ferrous or ferric condition, is non-toxic when taken orally or administered parental. The limit test of iron is based on reaction of iron in ammonical solution, in presence of citric acid with Thioglycolic gives purple colour. The colour produce by test solution is compared with standard iron solution (ferric ammonium sulphate). Citric acid form a soluble complex with iron and prevent precipitation by ammonium as ferrous hydroxide. Ferrous thioglycolate is colorless in nature & acid solution. The colour develop only in presence of alkali. It is unstable in presence of air due to oxidation to the ferric compound; therefore colour should be compared immediately after the time allowed for food development of colour is over. Thioglycolic acid also reducing agent, hence any ferric ions present are converted to ferrous ions, which form thioglycolate. 73

74. PRINCIPLE………(Continue) It is believed that the thioglycolic acid that has been more sensitive than the ammonium thiocynate test. Hence, ammonium thiocynate is replaced by thioglycolic acid. Interferences of other metal cation is eliminate by making the use of 20% citric acid, whic forms a complex with other metal cation. Limit test of Iron is based on the reaction of iron in ammonical solution with thioglycollic acid in presence of citric acid to form iron thioglycolate, which is pale pink to deep reddish purple in color.) Earlier aamonium thiocyanate reagent was used for the limit test of iron. Since thioglycolic acid is more sensitive reagent, it has replaced ammonium thiocyanate in the test . 74

75. 75 Reasons: Citric acid helps precipitation of iron by ammonia by forming a complex with it. Thioglycolic acid helps to oxidize iron (II) to iron (III). Ammonia to make solution alkaline The color of the 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.

76. Test sample Standard compound 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 up to 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 76

77. REACTION (Purple colour) OR Fe +2 + 2 CH 2 COO - HS Fe (SCH 2 COO - ) -2 Iron ion Thioglycolic acid Ferrous thioglycolate OBSERVATION The purple color 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. Means; the intensity of colour produced by sample is less than that of standard solution colour, it passes the test. 77

78. LIMIT TEST FOR ARSENIC 78

79. S.N. Chemical Quantity Apparatus Quantity 1 Lead acetate solution (10% w/v) Pb (C 2 H 3 O 2 ) 2 Q.S. Arsenic apparatus (Gutzeit Apparatus) 02 2 Potassium iodide (KI ) 2 gm Beaker (100 ml) 02 3 Zinc (Zn ) 20 gm Glass Rod 01 4 HgCl 2 Paper Q.S. Stand 01 To perform limit test for Arsenic for given unknown sample 79 REAGENT PREPARATION Preparation of the test solution: The solution of water soluble substance is prepared with water and stanneted HCl AsT. The solution of substance such as metallic carbonates, which effervesces with acids, is obtained with brominated HCl AsT. The substances, which are insoluble, e.g.: BaSO 4 , bentonite or kaolin are diffused in water.

80. Stanneted Chloride solution : It is prepared by adding Stannous Chloride solution to an equal volume of HCl AsT, reducing the original volume by boiling and filtering through a fine-grain filter paper. Stannated Hydrochloric acid : It is prepared by adding 1 ml of stannous chloride solution AsT to 100 ml of HCl AsT. Preparation of standard arsenic solution (10 ppm As): Dissolved 0.330 g of arsenic trioxide in 5ml of 2 M sodium hydroxide and dilute to 250.0 ml with water. Dilute 1 volume of this solution to 100 volumes with water. Zinc : It is the granulated zinc which complies with the following additional test: -To 10 gm of the granulated zinc adds 15 ml of the stannous chloride solution AsT and 5 ml of 0.1 M potassium iodide. -Apply the general test but allow the reaction to continue for one hour. -NO visible stain should be produced on the mercuric chloride paper. -Repeat the test by adding 0.1 ml of standard arsenic solution (10 ppm As); a faint but distinct yellow stain is produced. 80

81. PRINCIPLE Arsenic is harmful due to its toxic nature. Pharmacopoeia method is based on ‘Gutzeit Method’. Concentration of arsenic beyond 0.01 mg/L in pollutant by the World Health Organization (WHO ). 81

82. The depth of yellow stain on mercuric chloride paper will depend upon the quality of arsenic present in the sample, which is compared with that of standard stain produced from known amount of arsenic. 82

83. 83 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 agents like potassium iodide. It is also called as Gutzeit test and requires special apparatus. Arsenic, present as arsenic acid (H 3 AsO 4 ) in the sample is reduced to arsenious acid (H 3 AsO 3 ) by reducing agents like potassium iodide, stannous acid, zinc, hydrochloric acid, etc. Arsenious acid is further reduced to arsine (gas) (AsH 3 ) by hydrogen and reacts with mercuric chloride paper to give a yellow stain. Substance + dil HCl -------------------------  H 3 AsO 4 (contains Arsenic impurity) Arsenic acid H 3 AsO 4 + Arsenic acid H 2 SnO 2 -------------------→ H 3 AsO 3 + H 2 SnO 3 Arsenious acid H 3 AsO 3 + 6[H] ---------------------→ AsH 3 + 3H 2 O Arsenious acid nascent hydrogen Arsine gas The depth of yellow stain on mercuric chloride paper will depend upon the quantity of arsenic present in the sample.

84. 84 When the sample 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.

85. 85 Use of stannated 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: H 2 S gas may be formed during the experiment as zinc contains sulphides as impurities. It gives black stain to HgCl 2 paper and so will interfere the test. Hence , gases evolved are passed through cotton wool plug moistened with lead acetate, where H 2 S 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.

86. 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. Reasons Lead acetate pledger or papers are used to trap any hydrogen sulphide , which may be evolved along with arsine. 86

87. SN Standard Test 1 A know amount of dilute arsenic solution is kept in the wide mouthed bottle of the apparatus. The test solution is prepared by dissolving specific amount in water and stannated HCl (arsenic free) and kept in a wide mouthed bottle.  2 To this solution 1 gm of KI, 5 ml of stannous chloride acid solution and 10 gm of zinc is added (all this reagents must be arsenic free) To this solution 1 gm of KI, 5 ml of stannous chloride acid solution and 10 gm of zinc is added (all this reagents must be arsenic free) 3 Keep the solution aside for 40 min Keep the solution aside for 40 min 4 Compare the stain obtained on mercuric chloride paper with standard solution. Compare the stain obtained on mercuric chloride paper with standard solution. PROCEDURE Take 250 ml of the arsenic LT apparatus bottles. Labelled one is ‘Test’ and other is ‘standard’. 87

88. Regular arsenic apparatus with alternate device (a) and (b) for fixing mercuric chloride paper. Diagram 88

89. Assembly for Limit Test of Arsenic Sample Preparation Preparation of Mercuric Paper Standard (S) & Test (T) Attachment of Paper During Reaction Yellow stain observed after reaction completion Answer for How to Performed? 89

90. OBSERVATION If the stain produced by test is no deeper than standard stain, then sample complies limit test for arsenic.   CONCLUSION After 40 minutes, if the intensity of the yellow stain produced in the standard is more that in the test, the sample complies with the limit test of arsenic. RESULT Limit test of arsenic passes the test. 90

91. LIMIT TEST FOR HEAVY METAL 91

92. Limit test for heavy metals 92 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 In substances the 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 . These remain distributed in colloidal state, and give rise to a brownish coloration.

93. I.P limit for 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 : T h e me t hod i s appl i cab l e for the sampl e s which DO NOT give clear colourless solutions under specified conditions of test. Method III : Used for substances which give clear colourless solut i ons i n sodium hydroxide medium. 93 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.

94. LIMIT TEST FOR LEAD 94

95. Limit test for lead 95 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 thiocabazone (dithizone) in alkaline solution to form lead dithizone complex which is red in color. Dithizone in chloroform, 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.

96. 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. 96

97. Method: Sample solution is transferred to a separating funnel. To it 6 ml of ammonium citrate, 2 ml potassium cyanide and 2 ml of hydroxalamine HCl are added. 2 drops of phenol red Solution is made alkaline by adding ammonia solution. This is then extracted with 5 ml portions of dithizone solution until it becomes green. The combined dithizone extracts are shaken for 30 seconds with 30 ml of nitric acid and chloroform layer is discarded. To the acid solution 5 ml of standard dithizone solution is added and 4 ml ammonium cyanide and solution is shaken for 30 sec. Similarly prepare standard. 97

98. Observation: The intensity of 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. 98 Reasons: Ammonium hyd r ochloride , citrate, potassium cyanide, hyd r oxylamine i s u s ed to make pH optimum s o int e rference a n d 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.

99. A known quantity of 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 Esha Shah 99

100. Aq. Ammonia is added in limit test of lead: Pb+ S-------------  PbS In limit test of lead, PbS is produced by addition of standard H 2 S , 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. 100

101. LIMIT TEST FOR MODIFIED LIMIT TEST 101

102. MODIFIED LIMIT TEST FOR CHLORIDES 102 Depending upon the nature of the substance, some modifications have to be adopted for the preparation of the solution. Alkaline substances have to be dissolved in acid so that effervescence ceases and much of the free acid is left in the solution as is prescribed in the test. Insoluble substances are generally extracted with water and then filtered, and the filtrate is used for the test, because the presence of insoluble substance modifies the opalescence and colour.

103. Modified limit test for Chlorides Esha Shah 103 Salts of organic acids like sodium benzoate, sodium salicylate, etc. liberate free water insoluble organic acid during acidification which is filtered off and the filtrate is employed for the test. Coloured substances like crystal violet, malachite green, etc. are carbonised and the ash so produced is extracted in water. Deeply coloured substances have to be decolourised before test e.g., potassium permanganate is reduced by boiling with alcohol and the filtrate is used. Reducing substances like hypophosphorus acid, which react with silver nitrate in the limit test for chlorides should be oxidized with nitric acid or some other oxidizing agents before carrying out the test.

104. 104 Aim : To perform the limit test for chloride in potassium permanganate sample (according to IP’96 ) Requirement : Nessler’s cylinder, measuring cylinder, pipette, spatula, distilled water, dilute nitric acid, 0.1 M silver nitrate solution, potassium permanganate sample Principle : The limit test for chloride based on the reaction between soluble chloride impurities present in the substance and silver nitrate solution to give white precipitates of silver chloride. These white precipitates are insoluble in dilute nitric acid and hence give turbidity or opalescence to the test solution. The extent of the turbidity produced depends upon the amount of the chloride present in the substance which is compared with a standard opalescence produce by addition of silver nitrate to a standard solution having known amount of chloride and the same volume of the dilute nitric acid as the use in the test solution. Modified limit test for Chlorides

105. When potassium permanganate solution is treated with ethanol in presence of heat the redox reaction will take place, i.e. potassium permanganate gets reduced to manganese dioxide (precipitates). The filtrate of the reaction is colorless that is subjected to proceed for limit test for chloride. 105 If the turbidity developed in the sample is less than the standard turbidity, the sample passes the limit test for chloride and vice-versa. As potassium permanganate gives purple color aqueous solution that interferes in the comparison of opalescence or turbidity, therefore the aqueous solution must first be decolorized. Potassium permanganate is oxidizing agent while ethanol is reducing agent.

106. Chemical Reaction: 2 KMnO 4 + 3 C 2 H 5 OH----------  2 MnO 2 + 2 KOH + 2 CH 3 CHO + 2 H 2 O 106 S. N. STANDARD SOLUTION S.N. TEST SOLUTION 1 Take 10 ml chloride standard solution (25 ppm chloride) and add 5 ml water in a Nessler’s cylinder. 1 Transfer the prepared test solution in Nessler’s cylinder 2 Add 10 ml of dilute nitric acid and dilute to 50 ml with distilled water 2 Add 10 ml of dilute nitric acid and dilute to 50 ml with distilled water 3 Add 1ml of 0.1 M silver nitrate solution and stir immediately with glass rod and allow standing for 5 minutes protected from light. 3 Add 1ml of 0.1 M silver nitrate solution and stir immediately with glass rod and allow standing for 5 minutes protected from light. Compare the turbidity or opalescence produced in test solution with respect to standard solution and report the result and conclusion.

107. Observation and conclusion: Observation and conclusion will be of two types: If the intensity of turbidity or opalescence appears to be more in test solution than the standard solution then conclusion is impurities of chloride in given sample is over the limit as per IP’96. Hence, sample do not passes the limit test for chlorides. If the intensity turbidity or opalescence appears to be less or equal in test solution than the standard solution then conclusion is impurities of chloride in given sample is under the limit as per IP’96. Hence, sample passes the limit test for chloride 107

108. To Perform The Limit Test For Sulphate In Potassium Permanganate Sample (According To IP’96) 108 Principle : It is a comparison method. It involves the comparison of opalescence or turbidity of test sample verses standard sample which contain the definite amount of sulphate impurities. The limit test of sulphate is performed on the basis of reaction between the barium chloride reagent (containing barium chloride, sulphate free alcohol and solution of potassium sulphate (K 2 SO 4 ) and soluble sulphate in the sample with formation of barium sulphate (BaSO 4 ) white precipitates. Sulphate free alcoholic potassium sulphate is added to increase the sensitivity of the test. Very small amount of barium sulphate present in the reagents acts as a seeding agents for precipitation of barium sulphate, if sulphate is present in the sample under the test.

109. 109 Ethanol is added to prevent the super saturation i.e. the crystallization of sulphate with any other ion. As potassium permanganate gives purple colored aqueous solution that interferes in the comparison of opalescence or turbidity, therefore it requires to be decolorized. Potassium permanganate is oxidizing agent while ethanol is reducing agent. When potassium permanganate solution is treated with ethanol in presence of heat the redox reaction takes place, i.e. potassium permanganate gets reduced to manganese dioxide (precipitates) and ethanol gets oxidized to form ethanal . The filtrate of the reaction is colorless that is subjected to proceed for limit test for sulphate. Reaction 2 KMnO 4 + 3 C 2 H 5 OH 2 MnO 2 + 2 KOH + 2 CH 3 CHO + 2 H 2 O

110. Sr. No. STANDARD SOLUTION SR. NO TEST SOLUTION 1 Take 1 ml 25% w/v barium chloride in Nessler’s cylinder and add 1.5 ml of ethanolic sulphate standard solution (10 ppm SO 4 -2 ). Mix and allow to stand for 1 minutes 1 Take 1 ml 25% w/v barium chloride in Nessler’s cylinder and add 1.5 ml of ethanolic sulphate standard solution (10 ppm SO 4 -2 ). Mix and allow to stand for 1 minutes 2 Add 15 ml of standard sulphate solution (10 ppm SO 4 -2 ) and 0.15 ml of 5M acetic acid. 2 Transfer prepared test solution and add 0.15 ml of 5 M acetic acid. 3 Add sufficient distilled water to produced 50 ml. Stirred it immediately and allow standing for 5 minutes. 3 Add sufficient distilled water to produced 50 ml. Stirred it immediately and allow standing for 5 minutes. 110 Compare the turbidity or opalescence in the test solution by viewing transversely both solutions against black background .