ANALYST, AUGUST 1985, VOL. 110 985

Assay of pAminobenzoic Acid with Aromatic A/-Haloamines

Beby Jayararn Department of Chemistry, V. V. Pura College of Science, Bangalore-560 004, India and NetkaI M. Made Gowda Division of Biochemistry, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550, USA

The organic sulphonyl haloamines chloramine-T, bromamine-T and bromamine-B were used for the determination of p-aminobenzoic acid in aqueous solution. The proposed back-titration methods, which are based on the oxidation of p-aminobenzoic acid, are simple, fairly rapid and reproducible with an error of +I%. The reactions involve a four-electron change and the oxidation products were identified. The effects of pH and the presence of some foreign ions, and also pyridoxine, pyridoxalamine, glycerol and alcohol, on the oxidation were studied. Keywords: p-Aminobenzoic acid assay; organic sulphon yl haloamines; back-titration

Although modern trends in analytical techniques lean towards sophistication, the volumetric determination of s~bstratesl-~ with oxidising agents is still widely used because of its simplicity. The chemistry of aromatic sulphonyl haloamines:

0 Na R = H or CH3

o x X = CI or Br

has evoked interest as they are sources of halonium cations, hypohalite species and N-anions. They interact with a wide range of functional groups, effecting an array of molecular transformations. Considerable progress has been made in analytical chemistry with the introduction of some aromatic sulphonyl haloamines as redox titrants.lTsfj Although the toluene analogues of haloamines are well known, there is little information about the benzene derivatives.

Several methods have been developed for the determina- tion of p-aminobenzoic acid (PABA), which is pharmaceutic- ally important .7 A few direct titrimetric procedures are known for the assay of PABA by using sodium chlorite,B N-bromo- succinimidez or a bromate - bromide mixture.3 PABA has also been assayed by colorimetric,9 conductimetric,lO spectropho- tometric11 and chromatographic12 methods. However, some of these methods involve tedious experimental procedures. In this paper we report titrimetric methods developed for the assay of PABA using chloramine-T (p- CH3C6H4SO2NC1Na.3H20; abbreviated to CAT), bromamine-T (p-CH3C6H4So2NBrNa.3H20; abbreviated to BAT) and bromamine-B (C6H5S02NBrNa. 1 .5H20; abbre- viated to BAB) as oxidimetric reagents. The proposed methods are simple, rapid and reproducible.

Experimental Reagents

p-Aminobenzoic acid. PABA (Widner, UK) was used without further purification. Spectrophotometric assay13 showed the purity to be 99.9%. An aqueous solution of PABA (1 mg ml-1) was prepared.

Buffer solutions. The following buffer solutions were prepared according to standard methods? pH 1 and 2 (hydrochloric acid + potassium chloride); pH 3 (citric acid + disodium phosphate); pH 4-6 (sodium acetate + acetic acid); pH 7-9 (sodium tetraborate + orthoboric acid + sodium chloride); and pH 10 (sodium hydrogen carbonate + sodium carbonate).

Preparation of Organic Haloamines Bromamine- T BAT was prepared15 from dibromamine-T. About 20 g of dibromamine-T were dissolved with stirring in 30 ml of 4 M sodium hydroxide solution at 25 k 2 "C and the resulting aqueous solution was cooled in ice. The pale yellow crystals of BAT that formed were filtered under suction, washed quickly with the minimum volume of cold water and dried over phosphorus pentoxide. The purity of the compound was checked by the iodimetric determination of its active bromine content (theoretical 24.5% Br; found 24.4% Br) and by recording its 1H (obtained on a Varian 60-MHz NMR spectrometer) and 13C (obtained on a Bruker WH 270-MHz NMR spectrometer) NMR spectra. These spectra were obtained in CDCL3 solvent using tetramethylsilane (TMS) as the internal standard.

1H NMR spectrum (6 relative to TMS): 2.4 (singlet corresponding to CH,); 7.8 (doublet for ortho-H); 7.4 (doublet for meta-H). The coupling constant J,,, is 8.0 Hz.

13C NMR spectrum (p.p.m. relative to TMS): 145.39 (C-1, carbon attached to S atom); 140.50 (C-4); 131.75 (C-2,6); 129.40 (C-3,5); and 23.0 (methyl carbon).

Bromamine-B BAB was prepared16 by dissolving about 32 g of dibromamine-B in 50 ml of 4 M sodium hydroxide solution with constant stirring at room temperature and cooling the resulting aqueous solution in ice. The pale yellow crystals of BAB that formed were filtered under suction, washed with the minimum volume of cold water and dried over anhydrous calcium chloride. The compound was recystallised from hot water at 50 "C. the purity was checked by iodimetric determination of the active bromine present in the compound (theoretical 28.03% Br; found 27.90% Br) and by recording its l3C Fourier transform (FT) NMR spectrum (obtained on a Bruker WH 270-MHz NMR spectrometer).

13C FT NMR spectrum: the spectrum, obtained in D20 solvent using TMS as the internal standard, showed signals (p.p.m. relative to TMS) at 143.38 (C-1, carbon attached to S atom), 134.30 (C-4, para to the hetero atom), 131.26 (C-2,6) and 129.31 (C-33).

Chloramine-T CAT (E. Merck) was purified by the method of Morris eta1.17

Solutions Approximately 0.05 N solutions of BAT, BAB and CAT were prepared in distilled water and standardised by the iodimetric

Publ

ishe

d on

01

Janu

ary

1985

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

07:3

6:01

. View Article Online / Journal Homepage / Table of Contents for this issue

986 ANALYST, AUGUST 1985, VOL. 110

method. If stored in an amber-coloured bottle, these solutions remain stable for several days.

Preliminary Studies Aliquots of PABA solution were added to known excesses of volumes of the oxidants in separate glass-stoppered Erlen- meyer flasks containing sufficient acid, base or buffer solu- tions (as in Table 1). The reaction mixtures were set aside for various intervals of time at room temperature (25 f 2 "C) with occasional shaking. The excess of the oxidant, left uncon- sumed in each instance, was determined iodimetrically by titration with standard sodium thiosulphate solution.

Based on the four-electron stoicheiometry obtained in the preliminary studies (Table 1) for the oxidation of PABA by the three N-haloamines, the following procedure for the determination of PABA in solution was developed.

Recommended Procedure Place an aliquot of the solution containing 0.5-25 mg of PABA and 25 ml of 0.05 N CAT, BAT or BAB solution in a titration flask. Add sufficient acid, base or buffer solution to give the required concentration (0.1 M acetic acid or pH 5 for CAT; 0.05-0.10 M sodium hydroxide solution for BAT or BAB). Allow the reaction mixtures to stand for about 30 min with occasional shaking. Rinse the walls of the flask with about 20 ml of water, add 30 ml of 2 M sulphuric acid and 10 ml of 10% potassium iodide solution and titrate the liberated iodine with standard sodium thiosulphate solution to a starch end-point ( Vl ml). Run a blank under identical conditions (V2 rnl). The amount of PABA ( x mg) in the sample is given by the following equation:

where N is the normality of the sodium thiosulphate solution. x = 34.285N( V2 - V1)

Results and Discussion A typical set of results for the extent of oxidation of PABA in different media is given in Table 1. A four-electron oxidation of PABA with CAT was observed in acidic solutions. Among the acids, acetic acid favoured the stoicheiometric reaction. With buffer solutions the stoicheiometric reaction was noted in the buffer of pH 5 in 30 min. The oxidation rate decreased with increase in pH. Oxidation of PABA with BAT and BAB was slow in mineral acids and in buffer solutions of pH 1-8.

Table 1. Extent of oxidation of PABA with N-haloamines in various media. Time, 30 min; temperature, 25 f 2 "C,; PABA, 10 ml of solution; oxidant or N-haloamine, 25 ml of 0.05 N solution

Moles of oxidant consumed/mole of PABA taken

Medium 0.1 MHCI

0.1 M HC104 0.1 M H2SO4

0.1 M CH3COOH p H 1 PH2 PH3 PH4 PH 5 PH6 PH7 PH 8 PH 9 pH 10 0.05 M NaOH 0.10 M NaOH

CAT 2.74 2.25 1.88 2.01 2.81 2.53 2.41 2.18 2.01 1.88 1.21 0.31 0.24 0.18 0.12 0.12

BAT 0.23 0.12 0.14 0.16 0.22 0.21 0.36 0.48 0.52 0.52 0.88 0.93 1.64 1.87 2.01 2.01

BAB 0.19 0.10 0.15 0.18 0.23 0.24 0.39 0.42 0.47 0.54 0.79 0.84 1.23 1.58 1.99 2.01

However, a four-electron stoicheiometric reaction of PABA with these oxidants was observed in alkaline solutions (0.05-0.10 M sodium hydroxide solution) in 30 min. The time dependence of the percentage oxidation of PABA under different experimental conditions is shown in Fig. 1, from which it is clear that a stoicheiometric oxidation including 100% completion of the reaction occurs in about 30 min.

A comparison of the coefficients of variation of the results (not presented) showed that the accuracy of determination of PABA with the N-haloamines decreased in the order CAT>BAT>BAB. The results of some typical analyses of PABA are presented in Table 2. A comparison is made with the colorimetric methods through the t-test. Values of t for CAT, BAT and BAB were 2.24, 1.48 and 3.35. From statistical tables18 for n = 4, the confidence limits are set at 95 and 99% probability. It is found that = 3.18 (at 95% probability) and t0.995 = 5.84 (at 99% probability). Hence the difference between the colorimetric and CAT and BAT methods is not significant at both the 95% confidence levels. On the other hand, with BAB t = 3.35 and hence the difference is significant at the 95% level. The observed four-electron stoicheiometry for the PABA oxidation in acidic and alkaline media can be represented by the following equations: C6H4(NH2)COOH + 2RNXNa + 2H+ -+

C6H4(NH2)C00- + 2RNXNa + 2H20 -+

(2) where R = p-CH3C6H4S02- and X = C1 for CAT and Br for BAT, and R = C6H5S02- and X = Br for BAB.

C6H2X2(NH2)COOH + 2RNH2 + 2Na+ . . . . (1)

C6H2X2(NH2)C00- + 2RNH2 + 2Na+ + 20H-

V I I I 1 I

0 5 10 15 20 25 30 Ti me/m in

Fig. 1. Variation of the percentage of oxidation of PABA by different N-haloamines with time. 1, Chloramine-T; 2, bromamine-T; and 3, bromamine-B. Media: l,O.l M CH3COOH; 2, 0.05 M NaOH; and 3, 0.1 M NaOH. PABA, 1 mg ml-1 solution; N-haloamine, 25 ml of 0.05 N solution

Table 2. Determination of PABA with organic haloamines. Values from the spectrophotometric13 method are given in parentheses

PABA foundlmg

PABA taken/mg 0.53 1.25 2.57 5.33 7.15

10.52 15.75 18.36 20.55 25.39

CAT 0.53 (0.53) 1.27 2.59 (2.56) 5.31 7.14 (7.14)

10.54 15.78 18.34( 18.36) 20.59 25.36

BAT 0.52 1.27 2.55 5.35 7.18

10.50 15.72 18.33 20.51 25.41

BAB 0.52 1.24 2.55 5.31 7.18

10.49 15.72 18.39 20.51 25.34

Publ

ishe

d on

01

Janu

ary

1985

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

07:3

6:01

. View Article Online

ANALYST, AUGUST 1985, VOL. 110 987

The substrate, PABA, contains a weak electrophilic -COOH group and a strongly nucleophilic (or electron- releasing) -NH2 group, which is an ortho- and para-director in electrophilic aromatic substitution reactions. Each haloamine contains an electrophilic (or electron-withdrawing) halonium, X+, ion. Therefore, the reaction between PABA and the haloamine involves an electrophilic aromatic substitution. Here X+ ion attacks the available ortho positions (with respect to the amino group) in the aromatic ring of PABA to form the product 3,5-dihalo-4-aminobenzoic acid. These halogen deri- vatives of PABA were isolated and identified from melting- point data (dichloro derivative, 293 “C; dibromo derivative, 330 “C).

The presence of p-toluenesulphonamide formed in the reaction with CAT and BAT was detected19 by TLC. Benzenesulphonamide obtained in the reaction with BAB was identified20 by a chromatographic method.

Detailed investigations of PABA indicated the following: 1. The common ions K+, Na+, Ba2+, Zn2+, C104-, S042-

and P043- (up to an ionic strength of 0.2 M) do not interfere in the assay.

2. Pyridoxine, pyridoxalamine and glycerol will interfere in the method.

3. The stoicheiometry of oxidation is unaffected by a reversal of the order of addition of the oxidant and PABA.

The authors are grateful to the Bangalore NMR facility, Indian Institute of Science, Bangalore, India, for the NMR spectra.

References 1 . Mahadevappa, D. S., Rangappa, K. S., Gowda, B. T., and

Gowda, N. M. M., Microchem. J., 1982, 27, 254, and references cited therein. Barakat, M. Z., Fayzalla, A. S., and El-Aassam, S., Micro- chem. J . , 1973 18,308.

2.

3. Ganapathy, K. , Ramanujam, M., and Neelakatan, K., Acta Cienc. India, Ser. Chem., 1978, 5 , 36.

4. Jayaram, B. , and Mayanna, S. M., Talanta, 1983,30, 798. 5. Campbell, M. M., and Johnson, G., Chem. Rev., 1978,78,65. 6. Bishop, E., and Jennings, V. J., Talanta, 1958, 1, 197. 7. Windholz, M., “The Merck Index, An Encyclopedia of

Chemicals and Drugs,” Ninth Edition, Merck, Rahway, NJ, 1976, p. 427.

8. Albert, F. M., Cimpu, V., Valeanu, M., and Jercan, E. R., Rev. Roum. Chim., 1966, 11, 1443; Chem. Abstr., 1967, 66, 101452s.

9. Eckert, H. W., J. Biol. Chem., 1943, 148, 197. 10. Preti, C., and Tosi, G., Anal. Chem., 1981, 53, 48. 11. Zajac, M., and Ksiezniakiewicz, B., Farm. Pol., 1972,28,959;

Chem. Abstr., 1973, 78,47857k. 12. Chaing, H. C., Lin, Y., and Wu, Y. C., J. Chromatogr., 1969,

45, 161. 13. Hashmi, M. H., “Assay of Vitamins in Pharmaceutical

Preparations,” Interscience, New York, 1973, p. 417. 14. Findlay, A., “Practical Physical Chemistry,” Longman, Lon-

don, 1954, p. 268. 15. Nair, C. G. R., Kumari, R. L., andsenan, P. I., Talanta, 1978,

25, 525. 16. Ahmed, M. S. , and Mahadevappa, D. S., Talanta, 1980, 27,

669. 17. Morris, J. C., Salazar, J. A, , and Wineman, M. A., J . Am.

Chem. SOC., 1948, 70, 2036. 18. Davis, 0. L., and Goldsmith, P. L., “Statistical Methods in

Research and Production,” Olivery & Boyd, Edinburgh, 1972, p. 438.

19. Mahadevappa, D. S. , and Gowda, N. M. M., Talanta, 1975,22, 771.

20. Yathirajan, H. S., Mahadevappa, D. S., and Swamy, R., Talanta, 1980, 27, 52.

Paper A41452 Received December 31st, 1984

Accepted March 4th, 1985

Publ

ishe

d on

01

Janu

ary

1985

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

07:3

6:01

. View Article Online