76 SRINIVASAN : POTENTIOMETRIC DETERMINATION OF SMALL AMOUNTS [VOl. 75 Potentiometric Determination of Small Amounts of Procaine, Sulphanilamide and Related Compounds Bromate - Bromide Titration BY K. R. SRINIVASAN SYNoPsIs-Determination of procaine by direct titration with potassium bromate solution, using a pair of polarised platinum electrodes, is described. The method is simple, rapid and yields results of high precision and accuracy. It is applicable to the determination of other local anaesthetics of the p-amino- benzoic ester type, and also of some of. the sulpha drugs. SMALL amounts of procaine, in ointments, tablets, blood, etc., have been determined by colorimetric methods1J0,1* after diazotisation of the prirnary amino-group and coupling with a suitable reagent to form a coloured compound.These methods are useful only within a limited working range of concentration; they require the use of special equipment and are not rapid and direct, as they need the preparation of standard reference curves or the use of internal standards. The A.O.A.C. method of determination of procaine by brominationle consists in hydrolysis to p-aminobenzoic acid, reaction with a large excess of bromine for 2 hours at room temperature and titration of the excess of bromine with thiosulphate solution after addition of potassium iodide, when, under these conditions, tribromoaniline is formed. This method is time- consuming, and unless the conditions are strictly followed, will lead to low recoveries owing to under-bromination.15 Day and TaggartS found that p-aminobenzoic acid is not satis- factorily detehined by the excess-bromine method because of precipitation of partially brominated products.This is due to the fact that while the velocity constant of formation of tribromoaniline from aniline, or of the dibromo-derivative of @-substituted anilines, is very high, being of the order of lo8 (sees), the rate of further substitution of the latter by bromine to form tribromoaniline by elimination of the substituent group in the $-position, is relatively low, the extent of this reaction depending on conditions of temperature,' time of contact with bromine and residual bromine excess.15 Satisfactory results are, however, to be expected in determinations by direct titration with bromate and bromide ifi acid solution, since the reaction here proceeds to and stops at the stage of dibromo-substitution. But the difficulty with bromometric titration lies in the determination of the end-point.The use of an external indicator such as starch-iodide paper renders the method tedious3; with irreversible internal indicators like methyl orange the titration has to be done very slowly and any local Concentration of bromine will cause the colour of the indicator to fade before the end-point; other indicators8 besides being slow, will not be useful in solutions that are coloured. These difficulties are obviated in the present method by the use of a pair of polarised platinum electrodes as described by Foulk and Bawden.6 A very sharp end-point is obtained, as the slightest excess of bromine in the solution at once depolarises the cathode and causes a large deflection of the galvanometer. DETERMINATION OF SULPHONAMIDES Sulphanilamide and its derivatives are determined with equal facility by bromometric titration.The British Pharmacopoeia2 method consists in titration of the acid solution of the drug at a low temperature (15" C.) with standard sodium nitrite solution, wherebyFeb., 19501 OF PROCAINE, SULPHANILAMIDE AND RELATED COMPOUNDS 77 the NH, group is diazotised. The end-point is indicated by the immediate formation of a blue colour with starch-iodide paste used as an external indicator. This titration is slow and tedious, and as the reaction slows down towards the end, one has to wait for a few minutes after each addition of the reagent before testing for the end-point.Further, some uncertainty as to the exact end-point is often experienced, for even before the theoretical amount of sodium Fig. 1. Titration Assembly nitrite has been added, a blue colour is formed within a few seconds of placing a drop of the solution on the starch iodide paste. Determination of sulphanilamide as its dibromo-derivative by the excess bromine methodll often leads to erratic results owing to over-bromination, the extent of which varies with temperature, time of contact with bromine and residual bromine exce~s,~ and check results are obtained only under strictly controlled conditions of the experiment. With sulphathiazole, sulphapyridine, sulphadiazine and sulphaguanidine, Wells16 found that the excess bromine method cannot be successfully applied for their determifiation without preliminary hydrolysis to sulphanilic acid by heating with hydrochloric acid under reflux.The direct method of titration with bromate as outlined here has the advantage of simplicity and rapidity over these methods, and accurate results are obtained even with small amounts (Table I). REAGENTS AND APPARATUS- bromate, dried at 120" C. for 1 hour, is dissolved in a litre of distilled water. with water. Potassium bromate solution, 0.01 N---0-2784 g. of Analytical Reagent grade potassium DiZuted hydrochZoric acid--230 ml. of concentrated acid (sp.gr. 1.18) is made up to a litre Potassium bromide solution-10 per cent.78 SRINIVASAN : POTENTIOMETRIC DETERMINATION OF SMALL AMOUNTS [VOl. 75 The electrode is made by fusing two short pieces (about half an inch) of platinum wire at the end of glass tubing of 3-mm. bore and 10-cm.length. Connections are made by means of copper leads, silver-soldered to the platinum wires. The copper leads are sleeved by two melting-point capillaries. A polarising voltage of about 10 millivolts is obtained from a 600-ohm resistor (I.R.C. Q watt) and a small spiral of Nichrome wire (No. 26 S.W.G.) having a resistance of about 3.11 ohms, connected in series across a 14-volt dry cell as shown in Fig. 1. G is a Leeds and Northrup type 2420 spot light galvanometer, In titrating very small quantities, a 5-ml. graduated pipette (N.P.L. certified class A accuracy) with a fine tip, operated by a hypodermic syringe and rubber tube, was used in the absence of a precision micro-burette. Stirring is done by any suitable stirrer or even by swirling the beaker by hand.PROCEDURE- An aliquot containing about 5 mg. of the substance is taken in a 30-ml. beaker; 1 ml. of the potassium bromide solution and 10 ml. of the diluted hydrochloric acid are added. The electrodes, which have been cleaned by means of hot chromic acid and washed with distilled water, are dipped into the solution and connected up in the circuit. The solution is titrated with the potassium bromate solution, the end-point being indicated by a permanent deflection of the galvanometer from the zero position. TABLE I DETERMINATION BY BROMATE TITRATION Substance Procaine hydrochloride Benzocaine . . Sulphanilamide . . Sulphapyridine .. Sulphaguanidine Sulphathiazole* Sulphadiazine* . . .. .. .. .. .. .. Sample weight, mg. .. 2.32 3.25 4.26 .. 2.72 1-86 3.88 .. 1.70 1.90 3.67 .. 3-76 2-78 4.56 .. 2.64 3-37 5.45 .. 2.54 1-84 4-5-9 .. 3.08 2.85 1-89 0.01 N KBrO, required, ml. 3-42 4.75 6.27 6-60 4-55 9-42 3.95 4.42 8.55 6.0 4.45 7-32 4.52 5.75 9.37 5.95 4-35 10.72 7.40 6.80 4-54 Amount found, mg* 2.33 3.24 4.27 2.726 1.88 3.89 1.70 1.91 3-675 3-74 2.775 4.56 2-625 3.34 5-44 2.53 1.85 4.66 3.08 2-835 1.892 Equivalent weight is one-sixth of the molecular weight. DISCUSSION Recovery, 100.4 99-6 100.3 100.6 101.0 100.2 100.0 100.5 100.2 99.5 99.8 100.0 9 9 4 99.1 99.8 99.6 100.0 99-3 100.0 99.5 100.1 % Table I gives the results of a few of the determinations made and shows the accuracy and reproducibility of the method, which are satisfactory, considering the small size of the sample used.All the samples were purified by recrystallisation from alcohol or water, dried, and weighed out by means of a micro-chemical balance. Only procaine hydrochloride and benzocaine were taken for investigation, although other local anaesthetics of the $-amino- benzoic ester type may be determined similarly. In all cases except sulphathiazole and sulphadiazine the equivalent weight was taken asFeb., 19501 OF PROCAINE, SULPHANILAMIDE AND RELATED COMPOUNDS 79 one-fourth of the molecular weight, as four equivalents of bromine are taken up according to the equation NH2 0 + 4Br -+ ’8. +2HBr X X where X represents the substituent group in the $-position with respect to NH,.With sulphathiazole and sulphadiazine, six equivalents of bromine are found to be required, presumably owing to bromine substitution in the thiazole and pyrimidine nuclei as weL4 The determinations were made a t the laboratory temperature of about 30°C. and appreciable deviations in the temperature of the solution were found to have a negligible effect on the final result. An acid concentration of about 2 N was maintained. The limitation of the method is that it is useful only when any of the substances con- sidered is present alone, and in the absence of other easily brominated compounds, e g . , phenols. APPLICATION TO PROCAINE PENICILLIN As the degradation products of penicillin obtained on treatment with acid consume bromine, it was necessary to separate the procaine from penicillin by extraction with chloroform after liberating the free base with ammonia as described by Shaw.13 The procaine is taken up in hydrochloric acid from the chloroform solution and the acid solution is titrated with standard bromate.One ml. of 0.01 N potassium bromate is equivalent to 049mg. of procaine base. The results are given in Table 11. TABLE I1 DETERMINATION OF PROCAINE IN PROCAINE BENZYL PENICILLIN mg. ml. %t 25.0 16.95 39.9 The method was applied for estimating procaine in procaine benzyl penicillin. Sample* weight, 0.01 N KBrO, required, Procaine, 20.7 14-05 40-0 8.3 5-60 39.8 * Squibbs “Crysticillin” brand of procaine penicillin G was used. t The theoretical value is 40.12 per cent. of procaine. Grateful acknowledgment is made to Sri K.V. Sundaram Ayyar, Government Analyst, for his helpful suggestions and interest in the work. The author is indebted to Col. S. L. Bhatia, the Surgeon-General with the Government of Madras, for permission to publish this paper. REFERENCES 1. 2. 3. 4. 5. 6. Francis, A. W., Ibid., 1926, 48, 1632. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. - , Ibid., 1942, 25, 747. Bandelin, F. J., and Kemp, C . R., Ind. Eng. Chem., Anal. Ed., 1946, 18, 470. British Pharmacopoeia, 1948, p. 603. Day, A. R., and Taggart, W. T., Ind. Eng. Chem., Anal. Ed., 1926, 20, 546. English, J. P., et al., J . Amer. Chem. SOC., 1940, 68, 453. Foulk, C. W., and Bawden, A. T., Ibid., 1926, 48, 2045. Francis, A. W., and Hill, A. T., Ibid., 1924, 46, 2500. Hahn, F. L., Ind. Eng. Chem., Anal. Ed., 1942, 14, 571. Hoshall, E. M., J . Assoc. Off. Agr. Chem., 1939, 22, 748. Lapih-e, C., Anal. Chim. Acta, 1947, 1, 337. Martindale’s Extra Pharmaco+oeia, 22nd Ed., Vol. 11, 1945, p. 356. Methods of Analysis, Assoc. Off. Agr. Chem., 6th Ed., 1945, p. 697. Shaw, W. H. C . , J . Pharm. Pharmacol., 1949, 1, 514. Ting, K. S., et al., J . Lab. and Clinic. Med., 1949, 34, 822. Wells, E. H., J . Assoc. Ofi. Agr. Chem., 1942, 25, 541. LABORATORY OF THE GOVERNMENT ANALYST MADRAS 15, INDIA KING INSTITUTE, GUINDY, November, 1949