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  2. INTRODUCTION During the past four decades a several newer complex organic medicinal compounds have been proved of therapeutic value. Evidently, these compounds posed two vital problems during their analysis (a) poor solubility (b) weak reactivity in aqueous medium
  3. Such substances which give poor end point due to being weak acids or bases in aqueous solutions can be analysed by non-aqueous titrations. Additionally the substances which are insoluble in water dissolve in organic solvents and can be titrated in non-aqueous media.
  4. Nonaqueous titration is the titration of substances dissolved in no aqueous solvent. It is suitable for the titration of very weak acids and weak bases.
  5. Weak bases which have Kb values less than 10–6 can not be assayed accurately in aqueous medium. However such weak bases can be titrated satisfactorily by non-aqueous titrations.
  6. Consider assay of a weak base In aqueous solution a very weak base may act as a weaker proton acceptor compared to water and cannot be titrated properly with distinct end point. However this difficulty can be overcome using non- aqueous solvents….
  7. In non-aqueous solvents like glacial acetic acid, it can accept proton readily when perchloric acid is used as titrant. HClO4 + CH3COOH CH3COOH2 + CH3COOH2 + + B BH+ + CH3COOH Similarly, substances which are weakly acidic in nature like phenol, barbiturates can be satisfactorily titrated in dimethylformamide solvent and using alkali methoxide as titrant.
  8. Non–aqueous solvents are classified into four groups A) Aprotic B) Protophilic C) Protogenic D) Amphiprotic
  9. A) Aprotic solvents These are chemically neutral and unreactive substances having low dielectric constant and don’t cause ionization of solutes and do not undergo reactions with acids and bases e.g. toluene and carbon tetrachloride.
  10. B) Protophilic solvents These solvents possess high affinity for protons. In presence of such strongly protophilic solvents acidic strength of weak acids is enhanced and they become comparable to strong acids e.g. liquid ammonia, amines and ketones. This is called as levelling effect.
  11. C) Protogenic solvents Thse solvents are acidic in nature and readily donate protons.Due to their ability to donate portons they enhance strength of weak bases e.g. sulhuric acid, hydrogen fluoride.
  12. D) Amphiprotic solvents These solvents are slightly ionized. They possess protogenic as well as protophilic properties. Thus they donate as well as accept protons e.g. water, alcohol, weak organic acids. Ethanoic acid generally displays acidic properties and dissociates to produce protons CH3COOH CH3COO- + H+ In presence of perchoric acid (stronger acid) it accepts a proton CH3COOH + HClO4 CH3COOH2 + + ClO4 3 3
  13. LEVELLING SOLVENTS strongly protophilic or strongly protogenic solvents act as leveling solvents in non-aqueous titrations. In presence of strongly protophilic solvents, all acids act to be of similar strength.
  14. The converse occurs with strongly protogenic solvents which cause all the bases to act as if they were of similar strength. Solvents which act in this manner are levelling solvents. Strong bases are levelling solvent for acids.
  15. DIFFERENTIATING SOLVENTS Strong protogenic/protophilic solvents exert a levelling effect on weak bases/acids A strong protogenic solvent cannot differentiate between weak base and a very weak base as it forces both to accept proton .
  16. • A relatively weak protogenic solvent can differentiate between a weak base and slightly weaker base by causing the relatively more basic substance to act as a base while not affecting the weaker one. • Thus exerting a differentiating effect. • Similarly weak protophilic solvents differentiate between weak acid and slightly weaker acid.
  17. SELECTION OF SOLVENT Selection of a proper solvent is essential in non-aqueous titrations. Especially important factors are the basicity and the dielectric properties of the solvent. Increased basicity of the solvent enhances the acidic properties of a dissolved acid (sample). A low dielectric constant of the solvent depresses the ionisation and thereby enhances the acid strength or base strength of the sample to be assayed.
  18. ASSAY BY NON-AQUEOUS TITRATIONS (a) Acidimetry in Non-aqueous Titrations It can be further sub-divided into two heads namely : (1) Titration of primary, secondary and tertiary amines (2) Titration of halogen acid salts of bases.
  19. (b) Alkalimetry in Non-aqueous Titrations Titration of acidic substances
  20. Acidimetry in Non-aqueous Titrations (TITRATION OF WEAK BASES) TITRANT - Perchloric Acid : It is a very strong acid and when it is made to dissolve in acetic acid, acetic acid behaves as a base and forms an ‘onium ion’ after combining with protons donated by the perchloric acid.
  21. As the CH3COOH2 + ion can instantly donate its proton to a base, therefore, a solution of perchloric acid in glacial acetic acid, behaves as a strongly acidic solution.
  22. e.g. Pyridine, a weak base, when dissolved in acetic acid, acetic acid exerts its levelling effect and subsequently increases the basic characteristics of the pyridine. Therefore, it is practically feasible to titrate a solution of a weak base in acetic acid against a mixture of perchloric acid in acetic acid. Thus, a sharp end point is achieved which otherwise cannot be obtained when the titration is performed in an aqueous medium.
  24. For non-aqueous titrations of weak bases, the following four steps are usually taken into consideration, namely : (i) Preparation of 0.1 N Perchloric acid (ii) Standardization of 0.1 N Perchloric Acid (iii) Choice of Indicators (iv) Effect of Temperature on Assays
  25. PREPARATION OF 0.1 N PERCHLORIC ACID Procedure : Gradually mix 8.5 ml of perchloric acid to 900 ml of glacial acetic acid with vigorous and continuous stirring. Now add 30 ml acetic anhydride and make up the volume to 1 litre with glacial acetic acid and allow to stand for 24 hours before use.
  26. The acetic anhydride reacts with the water (approx. 30%) in perchloric acid and some traces in glacial acetic acid thereby making the resulting mixture practically anhydrous.
  27. STANDARDIZATION OF 0.1 N PERCHLORIC ACID - • Usually potassium hydrogen phthalate is used as a standardizing agent for acetous perchloric acid. • To 500 mg of potassium acid phthalate add 25 ml of glacial acetic acid and add few drops of 5% w/v crystal violet in glacial acetic acid as indicator. This solution is titrated with 0.1 HClO4. The colour changes from blue to blue green. • 1 ml of 0.1N HClO4 = 0.020414 gms of potassium acid Phthalate.
  28. PRECAUTIONS DURING PREPARATION OF PERCHLORIC ACID (a) Perchloric acid is usually available as a 70 to 72% mixture with water. It usually undergoes explosive decomposition and, therefore, it is available always in the form of a solution.
  29. (b) Conversion of acetic anhydride to acetic acid requires 40-45 minutes for its completion. It being an exothermic reaction, the solution must be allowed to cool to room temperature before adding glacial acetic acid to volume
  30. CHOICE OF INDICATORS Indicators commonly used in non-aqueous titrations are Thymol Blue, Thymolphthalein , Phenolphthalein Azo Violet ,O-nitroaniline. Following indicators are also widely used ….
  31. EFFECT OF TEMPERATURE ON ASSAYS Generally, most non-aqueous solvents possess greater coefficients of expansion as compared to water which is why small differences in temperature may afford significant and appreciable errors . Hence, it is always advisable to carry out standardization and titration preferably at the same temperature.
  32. EXAMPLES OF ACIDIMETRY 1) Titration of primary, secondary and tertiary amines Methyldopa Methacholine Chloride Adrenaline Chlordiazepoxide Quinine sulphate Salbutamol sulphate Sodium benzoate
  33. 2)Titration of Halogen Acid Salts of Bases- In general, the halide ions, namely chloride, bromide and iodide are very weakly basic in character so they cannot react quantitatively with acetous perchloric acid.
  34. To overcome this problem, mercuric acetate is usually added to a halide salt thereby causing the replacement of halide ion by an equivalent amount of acetate ion, which serves as a strong base in acetic acid e.g. Amitriptyline Hydrochloride Ephedrine hydrochloride Lignocaine hydrochloride
  35. Assay of Ephedrine HCl
  36. PRINCIPLE • Non-aqueous acid base titration • Very weakly basic • Can not react quantitatively with acetous perchloric acid • Hence Mercuric acetate is added to replace halide ion with an equivalent quantity of acetate ion, which is a strong base in acetic acid Assay of Ephedrine HCl
  37. ASSAY PROCEDURE : Weigh accurately about 0.17 g of Ephedrine Hydrochloride, dissolve in 10 ml of mercuric acetate solution, warming gently, add 50 ml of acetone and mix. Titrate with 0.1 M perchloric acid, using 1 ml of a saturated solution of methyl orange in acetone as indicator, until a red colour is obtained. Carry out a blank titration. Subtract blank from sample reading. Calculate percent purity of the sample.
  38. Ephedrine HCl assay reaction
  39. Factor for assay of Ephedrine HCl 201.7 gms ≡ 1000 ml 1 M HClO4 0.02017 gms C10H15NO,HCl ≡ 1 ml 0.1 M HClO4
  40. Standardization of HClO4 To 500 mg of potassium acid phthalate add 25 ml of glacial acetic acid and add few drops of 5% w/v crystal violet in glacial acetic acid as indicator. This solution is titrated with 0.1 HClO4. The color changes from blue to blue green.
  41. Assay of Ephedrine HCl
  42. Assay of sodium benzoate
  43. Preparation and standardization of titrant 0.1N solution of HClO4 Dissolve 8.5 ml of 72% HClO4 in about 900 ml glacial acetic acid with constant stirring, add about 30 ml acetic anhydride and make up the volume (1000 ml) with glacial acetic acid and keep the mixture for 24 hours. Acetic anhydride absorbs all the water from HClO4 and glacial acetic acid and renders the solution virtually anhydrous. HClO4 must be well diluted with glacial acetic acid before adding acetic anhydride because reaction between HClO4 and acetic anhydride is explosive.
  44. Assay Procedure : Weigh accurately about 0.25 g of Sodium Benzoate, dissolve in 20 ml of anhydrous glacial acetic acid, warming to 50º if necessary, cool. Titrate with 0.1 M perchloric acid, using 0.05 ml of 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01441 g of C7H5NaO2.
  45. ALKALIMETRY IN NON-AQUEOUS TITRATIONS Weakly acidic pharmaceutical substances may be titrated effectively by making use of a suitable non- aqueous solvent with a sharp end-point. such organic compounds include…. Anhydrides, Acids, Amino Acids, Acid Halides.
  46. Titrants used- 0.1 N Potassium Methoxide in Toluene-Methanol Sodium Methoxide Lithium Methoxide Tetrabutylammonium Hydroxide Primary standard : Benzoic acid Indicator: Thymol blue Dimethylformamide (DMF) is used as solvent
  47. Standardization of 0.1 N Methoxide Solution • Procedure:. Transfer 10 ml of DMF in a conical flask and add to it 3 to 4 drops of thymol blue and first neutralize the acidic impurities present in DMF by titrating with 0.1 N lithium methoxide in toluene-methanol. Quickly introduce 0.06g of benzoic acid and titrate immediately with methoxide in toluene-methanol. • Caution: Care must be taken to avoid contamination of neutralized liquid with atmospheric carbon dioxide.
  48. • The clear solution of sodium methoxide must be kept away from moisture and atmospheric CO2 as far as possible so as to avoid the following two chemical reactions that might ultimately result into the formation of turbidity. H2O + CH3ONa → CH3OH + NaOH H2CO3 + 2CH3ONa → 2CH3OH + Na2CO3
  49. Assay of Ethosuximide • Procedure: Weigh accurately about 0.2 g of the sample, dissolve in 50 ml of dimethylformamide, add 2 drops of azo-violet solution and titrate with 0.1 N sodium methoxide to a deep blue end point, taking precautions to prevent absorption of atmospheric carbon dioxide. Perform a blank determination and make any necessary correction. • Each ml of 0.1 N sodium methoxide is equivalent to 0.01412 g of C7H11NO2 .
  50. Reaction involved in Ethosuximide Assay
  51. Examples of drugs assayed by alkalimetry  AcetazolamideEthosuximide  Ethosuximide  Allopurinol  Nalidixic Acid  Diloxanide Furoate  Hydrochlorothiazide