Iodimetric Assays

IODIMETRIC ASSAYS

In such estimations, the pharmaceutical substances can be measured either directly or back titration of excess iodine with sodium thiosulphate solution.

1. Direct Titration with Iodine

(a) Preparation of 0.1 Iodine Solution

Theory : Iodine in aqueous solution acts as an oxidizing agent which forms the basis of assay methods involving direct titration with iodine. Thus, we have :

Materials Required : Iodine : 3.2 g ; potassium iodide : 7.5 g.

Procedure : Weigh accurately 3.2 g of crushed iodine crystals on a watch glass and transfer to a beaker containing potassium iodide (7.5 g) and water (10 ml). Dissolve the contents of the beaker with the help of a glass rod and frequent swirling. Transfer the contents of the beaker quantitatively to a 250 ml volumetric flask and make up the volume with DW.

Explanation : Iodine is sparingly soluble in water but undergoes rapid dissolution in the presence of potassium iodide due to the formation of the corresponding triiodide ion :

I₂  +  I^– → I₃^–

Thus, potassium iodide plays dual role, viz., in iodimetry—to solubilize iodine in aqueous KI solution, and in iodometry—as reducing agent, the excess KI helps in retaining liberated I₂ in solution through interaction with KI.

(b) Standardization of 0.1 Iodine Solution with the aid of Arsenic Trioxide (As₂O₃)

Theory : This particular standardization is solely governed by the following equations, namely :

Hydroiodic acid (HI) possesses strong reducing characteristics which renders the oxidation with iodine into a reversible reaction as follows :

In order to shift the equilibrium to the right-hand-side (i.e., towards As₂O₅) in the above reaction, sodium bicarbonate (NaHCO₃) is employed to remove the HI generated. It is important to record here that neither sodium hydroxide nor sodium carbonate can be used as both of them produce sodium iodide (NaI) and sodium iodate (NaIO₃) as designated below :

Materials Required : Arsenic trioxide : 0.5 g ; sodium hydroxide solution (20% w/v in water) : 2 ml ; dilute hydrochloric acid (2N) ; sodium bicarbonate : 4 g ; 0.1 N iodine solution.

Procedure : Weigh accurately 0.5 g arsenic trioxide into a beaker, add to it 2 ml of sodium hydroxide solution, and heat to dissolve. Cool and transfer the contents quantitatively to a 100 ml volumetric flask and make up the volume upto the mark with DW. Pipette 20 ml into an iodine-flask, acidify with dilute HCl carefully and confirm it by adding a little NaHCO₃ to remove the free excess acid, followed by a further 2 g to get rid of HI formed in the reaction mixture. Now, titrate with 0.1 N iodine solution till the end-point is achieved by the appearance of the first permanent pale straw colour.

(c) Standardization of 0.1 Iodine Solution by the aid of Sodium Thiosulphate

Theory : Iodine solution may also be standardized by using sodium thiosulphate (AR-Grade) whereby the latter gets oxidized to sodium tetrathionate as expressed below :

Materials Required : Sodium thiosulphate (AR) : 6.025 g ; 0.1 N I₂ solution.

Procedure : Weigh accurately 6.025 g of sodium thiosulphate (AR) to a 250 ml volumetric flask. Dissolve it in DW, shake well and make up the volume to the mark with DW. Pipette 25 ml of 0.1 iodine solution into an iodine flask and titrate with the standard sodium thiosulphate solution (as primary standard) until the solution becomes almost colourless.

Note : Stock solutions of sodium thiosulphate may be preserved by the addition of a few drops of sodium hydroxide solution (20% w/v) which serves as stabilizer as well as prevents decomposition.

(d) Preparation of Starch Solution

Material Required : Starch (arrowroot) : 1.0 g.

Procedure : Weigh 1.0 g starch in a glass in a glass pestle-mortar and triturate thoroughly with 10 ml of cold DW. Boil separately 200 ml of DW in a beaker and add the starch paste to it with vigorous stirring. The resulting mixture is boiled gently for a further period of 30 minutes till a transluscent and thin liquid having an uniform consistency is obtained.

Note : (1) The prepared solution of starch undergoes rapid deterioration, hence it is always desired to use freshly prepared solution every day,

(2) It is now more or less believed that the iodine is held as an ‘absorption complex’ within the helical chain of the macromolecule β-amylose i.e., a component of most starches. However, another component, α-amylose, is undesirable because it produces a red-colouration with io-dine which is not readily reversible, and

(3) ‘Soluble Starch’ comprises principally of β-amylose, with the α-fraction having been removed. Always, it is a practice to prepare indicator-solutions from this product exclusively.

1.1. Analgin

Materials Required : Analgin : 0.4 g ; alcohol (95%) : 40 ml ; 0.01 N hydrochloric acid : 10 ml ; 0.1 N iodine solution.

Theory : The estimation of analgin depends upon the oxidation of the enolic group with iodine. The reaction is not reversible :

Procedure : Weigh accurately about 0.4 g and dissolve in a mixture of 40 ml of alcohol and 10 ml of 0.01 N hydrochloric acid. Titrate the resulting mixture with 0.1 N iodine solution till a yellow colour that remains stable for 30 seconds is achieved. Each ml of 0.1 N iodine is equivalent to 0.016670 g of C₁₃H₁₆N₃NaO₄S.

1.2. Acetarsol

Materials Required : Acetarsol : 0.25 g ; sulphuric acid (conc.) : 7.5 ml ; nitric acid (fuming) : 2.5 ml ; ammonium sulphate : 5 g ; potassium iodide : 1.0 g ; sodium sulphite (0.1 N) : 1.0 ml ; phenolphthalein solution : 2 drops ; NaOH solution (0.1 N) ; dilute sulphuric acid (6 N) ; sodium bicarbonate : 8.0 g ; iodine solution (0.1 N).

Theory : Acetarsol is an organic arsenal, hence arsenic may be estimated by carrying out the oxidation As³⁺ to As⁵⁺ state with the help of 0.1 N iodine solution.

The organic entity present in acetarsol is destroyed primarily by boiling it with aqua-regia (a mixture of conc. H₂SO₄ and fuming nitric acid). The resulting mixture is heated in the presence of ammonium sulphate to  get rid of nitric acid finally in the form of nitrous oxide (N₂O) as follows :

Previously added H₂SO₄ maintains an acidic medium which on adding KI liberates HI that reduces the As⁵⁺ to As³⁺ state. Reduction is completed by boiling the solution which also expels the liberated I

as shown below :

The resulting mixture is cooled to room temperature and the residual iodine is removed by titration with 0.1 N sodium sulphite solution. Now, the solution is treated with sodium hydroxide solution to make it alkaline and then acidified carefully with dilute H₂SO₄ to remove the free NaOH. Finally, the resulting solution is made alkaline with NaHCO₃ so that the equilibrium is shifted to the right (i.e., AS³⁺ gets converted to As⁵⁺) quantitatively on carrying out the titration with 0.1 N iodine solution. Thus, we have :

Procedure : Weigh accurately about 0.25 g of acetarsol into a 500 ml iodine flask and add to it sulphu-ric acid (conc.) 7.5 ml, followed by nitric acid (fuming) 1.5 ml. Boil the contents of the flask gently for 45 minutes preferably in a fume-cupboard. Cool the solution, add 0.5 ml of fuming HNO₃ and boil till brown vapours (N₂O) stop coming. Again cool the contents and add carefully 5 g of ammonium sulphate in small lots at intervals and heat till there is no evolution of N₂O thereby giving rise to a colourless liquid. Bring the solution to room temperature, dilute with 100 ml DW, add 1 g KI and heat gently till the volume becomes 50 ml. Cool and add a few drops of 0.1 N sodium sulphite to effect decolourisation. Add 60 ml DW to dilute the resulting contents and make it just alkaline with NaOH solution by adding phenolphthalein indicator. Finally, acidify with dilute H₂SO₄, neutralize with NaHCO₃ and add 4 g of NaHCO₃ in excess. Swirl the contents of the flask and titrate with 0.1 N iodine solution. Each ml of 0.1 N iodine solution is equivalent to 0.01375 g of C₈H₁₀AsNO₅.

1.3. Cognate Assays

The following pharmaceutical substances can be assayed by direct titration with iodine as stated in Table 7.1.

2. Residual Titration Method (Excess of Iodine Titrated with Sodium Thiosulphate)

In this titration method an excess of iodine solution is added to the solution of the substance and thus, the latter gets oxidized quantitatively. The excess of iodine is subsequently back titrated with sodium thiosulphate using freshly prepared starch solution as indicator with an end-point from violet to colourless.

2.1. Benzylpenicillin

Theory : Benzylpenicillin can be assayed efficiently by adopting the following three steps sequentially, namely :

Step 1 : Benzylpenicillin is first converted to the corresponding penicilloic acid (a dicarboxylic acid) by carrying out the hydrolysis with sodium hydroxide solution, as follows :

Step 2 : Penicilloic acid on treatment with acid yields D-penicillamine and benzylpenilic acid, as shown under :

Step 3 : D-Penicillamine thus obtained is oxidised quantitatively by iodine to give rise to a disulphide, as expressed in the following equation ; whereas, the excess iodine is back titrated with 0.02 N sodium thiosulphate solution :

From the above reaction, we have :

C₁₆H_l7N₂NaO₄S ≡ I ≡ e

In usual practice, however, benzylpenicillin sodium is standardised against a chemical reference sub-stance of pre-determined potency.

Materials Required : Benzylpenicillin : 0.1 g ; (N) sodium hydroxide solution : 5 ml ; buffer solution (5.44% w/v of CH₃COONa and 2.40%  w/v  of  glacial  acetic  acid) : 20 ml ; (N)  hydrochloric acid : 5 ml ; 0.02 N iodine solution : 25 ml ; 0.02 N sodium thiosulphate solution ; starch solution.

Procedure : Weigh accurately about 0.1 g of benzylpenicillin in DW and dilute to 100 ml in a volumet-ric flask. Transfer 10.0 ml to an iodine flask, add 5 ml of N sodium hydroxide and allow to stand for 20 minutes. Now, add 20 ml of freshly prepared buffer solution, 5 ml of N HCl and 25.0 ml of 0.02 N iodine solution. Close the flask with a wet glass-stopper and allow to stand for 20 minutes in a dark place (i.e., protected from light). Titrate the excess of iodine with 0.02 N sodium thiosulphate, employing freshly pre-pared starch solution as an indicator added towards the end-point.

To another 10.0 ml of the initial solution add 20 ml of the buffer solution, allow to stand for 20 minutes in the dark and titrate with 0.02 N sodium thiosulphate, using starch solution, added towards the end of the titration as indicator.

The difference between the two titrations represents the volume of 0.02 N iodine equivalent to the total penicillins present in the given sample of benzylpenicillin. An assay may be carried out simultaneously by benzylpenicillin sodium (reference sample) so as to determine the exact equivalent of each ml of 0.02 N iodine.

Calculations : Calculate the potency in Units of penicillin from the declared number of Units of pencillin in benzylpenicillin sodium (reference sample).

2.2. Sodium Metabisulphite

Theory : Sodium metabisulphite in acidic medium (HCl) yields SO₂ which reacts with water to pro-duce sulphurous acid. The generated sulphurous acid is quantitatively oxidized by iodine to sulphuric acid, and the excess iodine is subsequently back titrated with sodium thiosulphate. The various reactions can be expressed as shown below :

Materials       Required :        Sodium       metabisulphite : 0.2 g ; 0.1 N Iodine solution ; hydrochloric acid ( ~−  11.5 N) : 1 ml ; 0.1 N sodium thiosulphate ; starch solution.

Procedure : Weigh accurately about 0.2 g of sodium metabisulphite and dissolve in 50.0 ml of 0.1 N iodine solution and add 1 ml hydrochloric acid. Titrate the excess of iodine with 0.1 N sodium thiosulphate employing freshly prepared starch solution, added towards the end of the titration, as indicator. Each ml of 0.1 N iodine is equivalent to 0.0047453 g of Na₂S₂O₅.

2.3. Cognate Assays

A few other pharmaceutical substances may also be assayed by adopting the residual titration method as shown in Table 7.2.