Assay by Non-Aqueous Titrations

ASSAY BY NON-AQUEOUS TITRATIONS

Assays of various pharmaceutical substances either in pure form or in dosage form may be assayed successfully by non-aqueous titrations. For the sake of convenience these typical titrations can be catego-rized into two broad groups, namely :

(a) Acidimetry in Non-aqueous Titrations—It can be further sub-divided into two heads, namely :

(i) Titration of primary, secondary and tertiary amines, and

(ii) Titration of halogen acid salts of bases.

(b) Alkalimetry in Non-aqueous Titrations—i.e., titration of acidic substances.

1. ACIDIMETRY IN NON-AQUEOUS TITRATIONS

In order to perform feasible titrations of weak bases, the solvent system should be selected specifically in such a fashion so as to eliminate as far as possible the competing reaction of water for the proton besides enhancing the strength of the basic species.

1.1. Titration of primary, secondary and tertiary amines

1.1.1.  Methlyldopa

In general, the reaction taking place between a primary amine and perchloric acid may be expressed as follows :

The specific reaction between methyldopa and perchloric acid is expressed by the following equation :

Materials Required : Methyldopa 0.2 g ; anhydrous formic acid : 15 ml ; glacial acetic acid : 30 ml ; dioxane : 30 ml ; 0.1 N perchloric acid and crystal violet solution.

Procedure : Weigh accurately about 0.2 g and dissolve in 15 ml of anhydrous formic acid, 30 ml of glacial acetic acid and 30 ml of dioxane. Add 0.1 ml of crystal violet solution and titrate with 0.1 N perchloric acid. Perform a blank determination and make any necessary correction. Each ml of 0.1 N perchloric acid is equivalent to 0.02112 g of C₁₀H₁₃NO₄.

Calculations : The percentage of methyldopa present in the sample is given by :

1.1.2.  Methacholine Clloride

Materials Required : Methacholine chloride : 0.4 g ; glacial acetic acid : 50 ml ; mercuric acetate solution : 10 ml ; 0.1 N perchloric acid and crystal violet solution.

Procedure : Weigh accurately about 0.4 g, previously dried and stored in a vacuum desiccator, and dissolve in 50 ml of glacial acetic acid, add 10 ml of mercuric acetate solution, one drop of crystal violet solution and titrate with 0.1 N perchloric acid to a blue-green end point. Perform a blank determination and make any necessary correction. Each ml of 0.1 N perchloric acid is equivalent to 0.01957 g of C₈H_l8ClNO₂.

Equation :

Mercuric acetate : It is essentially added to prevent the interference of the hydrochloric acid dis-placed through the formation of the relatively un-ionized HgCl₂, thereby making a predominant shift in the equilibrium so that the titrimetric reaction is quantitative.

Blank Titration : It is usually carried out to account for the possible reaction of atmospheric moisture with the titrant perchloric acid and also to check the titrant being employed to bring about the blue-green end-point.

Calculations : The percentage of methacholine chloride in the sample may be calculated by the following expression :

1.1.3.  Cognate Assays

Table 5.1, enlists the various cognate determinations using different indicators but employing the same titrant i.e., 0.1 N perchloric acid.

1.1.4.  Potentiometric Titrations

These non-aqueous titrations may also be carried out with the help of potentiometric titrations which technique shall be discussed at length elsewhere in this book.

It is always preferred to first ascertain the equivalence point of a given neutralization reaction potentiometrically (i.e., an instrumental method of analysis) ; and secondly, by selecting an appropriate indicator that will ensure the sharpest colour change for the least increment of volume of titrant added near the equivalence point.

In actual practice, however, there are quite a number of non-aqueous titrations of pharmaceutical substances either in pure or in dosage forms that can be successfully performed potentiometrically.

1.2. Titration of Halogen Acid Salts of Bases

In general, the halide ions, namely : chloride, bromide and iodide are very weakly basic in character so much so that they cannot react quantitatively with acetous perchloric acid. In order to overcome this problem, mercuric acetate is usually added (it remains undissociated in acetic acid solution) 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 as shown below :

1.2.A. Amitriptyline Hydrochloride

Materials Required : Amitriptyline hydrochloride : 1.0 g ; mercuric acetate ; crystal violet ; 0.1 N perchloric acid ; glacial acetic acid.

Procedure : Weigh accurately about 1.0 g and dissolve in 50 ml of glacial acetic acid, warming slightly, if necessary, to affect the solution. Cool, add 10 ml of mercuric acetate solution, two drops of crystal violet solution and titrate with 0.1 N perchloric acid to a green end-point. Perform a blank determination and make any necessary correction. Each ml of 0.1 N perchloric acid is equivalent to 0.03139 g of C₂₀H₂₃N. HCl.

Equations :

Calculations :

1.3. Cognate Assays

The following estimations of various pharmaceutical substances can also be carried out by the aforesaid procedure (Table 5.3) :

2. ALKALIMETRY IN NON-AQUEOUS TITRATIONS

A plethora of weakly acidic pharmaceutical substances may be titrated effectively by making use of a suitable non-aqueous solvent with a sharp end-point. The wide spectrum of such organic compounds in-clude : anhydrides, acids, amino acids, acid halides, enols (viz., barbiturates), xanthines, sulphonamides, phenols, imides and lastly the organic salts of inorganic acids.

However, a weak inorganic acid e.g., boric acid, can be estimated conveniently employing ethylenediamine as the non-aqueous solvent.

2.1. Preparation of 0.1 N Potassium Methoxide in Toluene-Methanol

Materials Required : Absolute methanol : 40 ml ; dry toluene : 50 ml ; potassium metal : 4 g.

Procedure : Add into a dry flask, a mixture of methanol (40 ml) and dry toluene (50 ml) and cover it loosely. Carefully add freshly cut pieces of potassium metal to the above mixture gradually with constant shaking. After complete dissolution of potassium metal, add enough absolute methanol to yield a clear solution. Toluene 50 ml is added with constant shaking until the mixture turns hazy in appearance. The process is repeated by the alternate addition of methanol and benzene until 1 litre of solution is obtained, taking care to add a minimum volume of methanol to give a visible clear solution.

2.1.1. Preparation of 0.1 N Sodiun Methoxide

It is prepared exactly in a similar manner as for 0.1 N Potassium Methoxide, using 2.3 g of freshly-cut sodium in place of potassium.

2.1.2. Preparation of 0.1 N Lithium Methoxide

It is prepared as for 0.1 N Potassium Methoxide, but using 0.7 g of lithium in place of potassium.

2.2. Standardization of 0.1 N Methoxide Solution

Materials Required : Dimethylformamide (DMF) : 10 ml ; thymol blue (0.3% in MeOH) ; 0.1 N lithium methoxide in toluene-methanol ; benzoic acid : 0.6 g.

Procedure : The apparatus shown in Figure 5.1, is employed for the standardization of 0.1 N methoxide solution. 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 0.06 g 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.

Equations :  The various equations involved in the above operations are summarized as stated below:

Interaction between sodium metal and methanol is an exothermic reaction and hence, special care must be taken while adding the metal into the dry solvent in small lots at intervals with adequate cooling so as to keep the reaction well under control.

The clear solution of sodium methoxide must be kept away from moisture and atmospheric CO₂ as far as possible so as to avoid the above two chemical reactions that might ultimately result into the formation of turbidity.

Step 1 : It shows the solution of benzoic acid (primary standard) in DMF,

Step 2 : It depicts ionization of sodium methoxide,

Step 3 : It illustrates the interaction between the solvated proton and the methylated ion.

In summing up, the net reaction in the process of standardization has been expressed. The interaction between the water in the solvent (DMF) and the titrant is equivalent to the volume of sodium methoxide consumed by DMF or may be considered as a blank determination.

2.2.1. Ethosuximide

Materials Required : Ethosuximide : 0.2 g ; dimethylformamide : 50 ml ; azo-violet (0.1% w/v in DMF) : 2 drops ; sodium methoxide 0.1 N.

Procedure : Weigh accurately about 0.2 g, dissolve in 50 ml of dimethylformamide, add 2 drops of azo-violet solution and tirate 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.

Equations :

2.3. Cognate Assays

The following determinations as stated in Table 5.4 may be carried out effectively by using 0.1 N sodium hydroxide either titrimetrically using an appropriate indicator or potentiometrically :

2.4. Tetrabutylammonium Hydroxide

The alkalimetry in non-aqueous titrations may also be carried out efficiently by using tetrabutylammonium hydroxide along with an appropriate indicatior.

2.4.1. Preparation of 0.1 N Tetrabutylammonium Hydroxide in Toluene-Methanol

Materials Required : Tetrabutylammonium iodide : 40 g ; absolute methanol : 90 ml ; silver oxide : 25 g ; dry toluene : 150 ml.

Procedure : Carefully dissolve 40 g of tetrabutylammonium iodide (Bu₄NI) in 90 ml of absolute methanol, add to it 20 g of finely powdered purified silver oxide and finally shake the mixture thoroughly for 1 hour. Centrifuge about 2-3 ml of the resultant mixture and test for iodide in the supernatant liquid. In case, it gives a positive test, add about 2 g more of silver oxide and shake for an additional period of 30 minutes. The said method may be repeated until the supernatant liquid obtained is completely free from iodide. The mixture thus obtained is filtered through a fine sintered glass filter and finally rinse the container with 3 portions, each of 50 ml of dry toluene. These washings may be added to the filtrate and the final volume is made upto 1 litre with dry toluene. The clear solution may be flushed with CO₂-free nitrogen for at least five minutes and duly protected from both CO₂ and moisture during storage.

Equation :

2.4.2. Standardization of 0.1 N Tetrabutylammonium Hydroxide

Materials Required : Benzoic acid : 60 mg ; dimethylbromide : 10 ml ; thymol blue solution (0.3% w/v in methanol) ; 0.1 N tetrabutylammonium hydroxide.

Procedure : Accurately weigh about 60 mg of benzoic acid into 10 ml of previously neutralized dimethyl formamide to the blue colour of thymol blue (3 drops) by titration against 0.1 N tetrabutylammonium hydroxide. Allow the benzoic acid to dissolve gradually and completely and titrate with 0.1 N tetrabutylammonium hydroxide preferably in an atmosphere of CO₂-free nitroaen.

Calculations :

2.4.3.  Chlorthalidone

Materials Required : Chlorthalidone : 0.3 g ; pyridine (dehydrated) : 50 ml ; 0.1 N tetrabutylammonium hydroxide.

Procedure : Weigh accurately about 0.3 g and dissolve in 50 ml of dehydrated pyridine. Titrate with 0.1 N tetrabutylammonium hydroxide, determining the end point potentiometrically and protecting the solution and titrant from atmospheric carbon dioxide throughout the determination. Perform a blank determination and make any necessary correction. Each ml of 0.1 N tetrabutylammonium hydroxide is equivalent to 0.03388 g of C_l4H_1lClN₂O₄S.

Equations :

2.4.4.  Cognate Assays

The following pharmaceutical substances may be assayed by employing tetrabutylammonium hydroxide either by using a suitable indicator titrimetrically or potentiometrically as given in Table 5.5.

The assay of the aforesaid pharmaceutical substances with tetrabutylammonium hydroxide is on a mole-for-mole basis. As these are monobasic acids in character, therefore, they react quantitatively in a non-aqueous media with the base titrant, employing typical acid-base indicators to detect the end-points.