Acta Chim. Slov. 2000, 47, 349-362. 349 UV STUDY OF INDOLE AND 3-ACETYLINDOLE IN PHOSPHORIC AND HYDROCHLORIC ACID SOLUTIONS B. S. Andonovski* and G. M. Stoj kovic Institute of Chemistry, “St. Cyril and Methodius University, P.O. Box 162, 91001 Skopje, Macedonia Received 13-03-2000 Abstract Protonation of indole and 3-acetylindole in phosphoric and hydrochloric acid solutions was studied by UV spectroscopy. We present pKa values obtained by the CVA (Characteristic Vector Analysis) method. From the data, pK a values for protonation were obtained by using the Hammett's equation: pK a = H0 + log[c(BH+)/c(B)]. The dissociation constants and solvent parameter m* were obtained by application of the Excess Acidity Method. The position of protonation is discussed. Key words: UV spectra, indole, 3-acetylindole, protonation, dissociation constants, Hammett Acidity Function Method, Excess Acidity Method, Characteristic Vector Analysis. Introduction 2,3-Benzopyrrole (indole) and 3-methylindole (skatole) are formed by decomposition of the L-tryptophan.1 It was suggested that indole (I) and skatole are formed in the presence of enzymes. In this case I is transformed in indolyl-3-sulfuric acid and indolyl-3-glucuronic acid.1 Some indole derivatives are plant hormones.2-6 For spectrophotometric determination of which property I and/or its derivatives, examination of the stability of the indoles in various acidic media is used as test for method validation.7-10 The results obtained from UV study of the protonation of I, indolyl-3-acetic acid, indolyl-3-propionic acid and indolyl-3-butyric acid in perchloric acid solutions11 and sulfuric acid solutions,13 3-methylindole, D-tryptophan, 3-acetylindole and indole-2-carboxylic acid in perchloric acid solutions12 and sulfuric acid solutions14 are reported. B. S. Andonovski, G. M. Stojkovič: UV Study of Indole and 3-Acetylindole in Phosphoric and… 350 Acta Chim. Slov. 2000, 47, 349-362. From the dissociation constants of the nine indole derivatives, values of acidity function (H) have been determined for sulfuric and perchloric acid in the concentration range from 0.1 to 12.0 mol dm-3, and from 0.1 to 6.0 mol dm-3 respectively,15 by means of the relation H = pKBH - log I . The dissociation constants (pKa values) of the other indole derivatives are determined using H values.16-22 By application of the Excess Acidity Method,23 pKa values for I and 1-methylindole are determinated.24 In the present, we studied the influence of solvent (phosphoric and/or hydrochloric acid solutions) upon UV spectra of I and 3-acetylindole (3-AI) was examined. For determine she pKa values, from she date obtained by analyzing she UV-spectra by any she characteristic vectors, it is possible to separate the medium effect from the larger changes due to the protonation. Experimental Stock solutions of I and 3-AI were prepared in a mixture of water and ethanol (1.0% v/v). More dilute solutions of I or 3-AI were prepared from solutions (by keeping the concentration of I (or 3-AI) constant), in different the concentrations of phosphoric acid (between 1.0 and 11.5 mol dm-3) and hydrochloric acid (between 1.0 and 8.0 mol dm-3). The concentration of I or 3-AI for UV-measurements was chosen in such a way that the absorbance had a value between 0.1 and 1.0 at the studied wavelength. The UV spectra of each solution were recorded between 400 - 190 nm on a Hewlett-Packard 8452A Diode Array Spectrophotometer, in 1 cm quartz cells. A solution of phosphoric or hydrochloric acid (of the same concentration as in the investigated solutions) was used as a blank. The measurements were performed 1 - 1.5 min after preparing the solution at 27.0 ± 0.20 C in termostated cell. From the absorbances on four selected experimental wavelengths, molar absorptivity (e) of B and BH+ were determinated (Table 1). B. S. Andonovski, G. M. Stojkovič: UV Study of Indole and 3-Acetylindole in Phosphoric and… Acta Chim. Slov. 2000, 47, 349-362. 351 Table 1.Wavelengths and she corresponding molar absorptivity for I (c = 3.1 10 mol dm-3 and 3-AI (c = 4.010-5 mol dm-3) in different solvents._____________ Compound Solvent c/ mol dm-3 l / nm 1 3 1 (log e / mol- dm cm- ) I HCl 3.5 8.0 210 (4.42) (3.93) 216 (4.55) (3.75) 222 (4.43) (4.03) 228 (3.90) (3.71) H3PO4 3.5 11.0 204 (4.37) (4.00) 210 (4.47) (4.03) 216 (4.54) (4.00) 222 (4.41) (3.87) 3-AI HCl 1.0 6.0 280 (3.91) (3.50) 300 (4.13) (3.81) 320 (3.76) (4.00) 340 (2.86) (4.03) H3PO4 3.5 11. 0 280 (3.85) (3.45) 300 (4.10) (3.84) 320 (3.80) (4.02) 340 (2.99) (3.98) Results and discussion 1.UV spectra of indole in water and phosphoric acid and hydrochloric acid solutions Since the indoles posses basic properties it is important to know exactly which ionic species are present under certain conditions. Kouzumi [25] has noted that in medium with pH <0.5 zwitterionic tautomers (II) may be present (see Sheme 1). It is known that UV spectrum of I in water exhibits four bands which result from p-$p* transitions [26]. In water solution she UV spectrum I shows absorption maximum at:196 nm (1Ba), log{e} = 4.31; 216 nm (1Bb), log{e} = 4.49; 270 nm (1La), log{e} = 3.74; 276 nm and 286 nm (1Lb), log{e} = 3.73 [15], [UV spectrum of I was assigned and interpreted using the FREE ELECTRON MODEL [26]]. Figure 1 shows ultraviolet spectra of I at different concentration of phosphoric acid solutions. B. S. Andonovski, G. M. Stojkovič: UV Study of Indole and 3-Acetylindole in Phosphoric and… 352 Acta Chim. Slov. 2000, 47, 349-362. Figure 1. Ultraviolet absorption spectra of indole (c = 3.110-5 mol dm-3-A-in water) as a function of phosphoric acid concentration. The concentration of phosphoric acid: (B)-6.5; (C)-7.5; (D)-8.5; (E)-9.5 and (F)-10.5 mol dm-3 (top to bottom, at X = 216 nm). In general, the solvents (perchloric acid solutions or sulfuric acid solutions) cause changes in the intensity of the 1Ba, 1Bb, 1La and 1Lb bands, in analytical concentration (above 1.0 mol dm-3). 11,13,On the other hand, in phosphoric acid solutions the changes in intensity of all four bands above analytical concentration 5.5 mol dm-3 (Fig.1.) are noted. The changes in the UV spectra of I observed in phoshoric acid are in agreement with those, which were observed in perchloric or sulfuric acid solutions.11,13 In Table 2 characteristic bands of the UV spectrum of IH+ are listed. Table 2. Experimental transitions in the UV spectra of IH Ion IH+ in HClO4 H2SO4 H2SO415 H3PO4 HCl solvents Transitions A/nm 1Lb 276 280 281 280 270 1La __ __ __ __ __ 240(sh) 240(sh) 238 240(sh) 240(sh) 1Bb 232 234 233 232 234(sh) 1Ba 204 202 __ 204 206(sh) (sh)-shoulder B. S. Andonovski, G. M. Stojkovič: UV Study of Indole and 3-Acetylindole in Phosphoric and… Acta Chim. Slov. 2000, 47, 349-362. 353 In phosphoric acid solutions at concentration higher than 5.5 mol dm-3, the solvent influences on the shift of the absorption curves of I and causes loss of the isosbestic points (Fig.1). Figure 2 shows six curves reconstituted from the “mean” curve and first vector, using CVA.27,28 Method of Characteristic Vector Analysis Characteristic vector analysis (sometimes referred to as “principal component analysis” or eigenvector analysis) is a method of separating independent factors for sets of multivariate response data. The method can be used empirically for estimating the number of independent factors contributing to the total variation observed in a family of UV spectra. If p independent factors are involved in generating the absorbance curve, the sample responses at each wavelength for a given concentration will be given by A1 = A1 + c 1v11 + c2v21 + ...+ cpVp1 A2 = A2 + c 1V12 + c2V22 + ..+ cpVp2 (1) Ar = Ar + c1V1r + c2V2r + ...+ cpVpr where the choice of A is arbitrary and the mean values of the absorbance seem to be a convenient choice. The v“s are characteristic vectors, and c”s are weighting coefficients. Ultraviolet spectra of I in solutions of different concentration of phosphoric acid >5.5 mol dm-3, show isosbestic points at 236 and 288 nm. For comparison, isobestic points of 238 and 288 nm in UV spectra of I in sulfuric acid solutions and perchloric acid solutions 234, 244 and 288 nm are obtained. The agrement of these results indicate that CVA method can be used for determination of isobestic points. Reeves [29] reported, that have only two characteristic vectors. The first vector can be used in CVA to obtain always accounted for 96% or more of the total variability. The physical-chemical meaning of the first vector associate with the effect of the protonation and second vector with the medium effect. By application the CVA the family ten UV spectra of indole in phosphoric acid solutions calculation of the first vector accounts for 97.5%, meaning that the protonation effect prevails the medium effects. B. S. Andonovski, G. M. Stojkovič: UV Study of Indole and 3-Acetylindole in Phosphoric and… 354 Acta Chim. Slov. 2000, 47, 349-362. Figure. 2.The reconstituted UVspectra of indole (c = 3.1 10-5 mol dm-3, A-in water) as a function of concentration phosphoric acid. From top to bottom: B-6.5 mol dm-3; C-7.0 mol dm-3; D-7.5 mol dm-3; E-8.5 mol dm-3 and F-9.5 mol dm-3. Two isosbestic points at 238 and 288 nm obtained in UV spectra of I in sulfuric acid solutions are in agreement with the results obtained in phosphoric acid solutions. The wavelengths of the isosbestic points obtained for I in perchloric acid solutions do not agree with those in phosphoric or sulfuric acid. In UV spectrum of I in a phosphoric acid solution with concentration 11.5 mol l-3 there are absorption maxima at 204 (1Ba), 232 (1Bb) and a broad absorption band at 280 (1Lb) nm (Fig.1). This absorption spectrum of IH+ was compared to the spectra of IH+ in perchloric acid solution and sulfuric acid solution (Table 2). It is possible therefore, that the protonation of I is leading to the 3-C atom (structure III), and IH+ cannot have structures II or IV (See sheme 1). B. S. Andonovski, G. M. Stojkovič: UV Study of Indole and 3-Acetylindole in Phosphoric and… Acta Chim. Slov. 2000, 47, 349-362. 355 H HH Scheme 1 UV spectra of indole in hydrochloric acid solutions The observed changes in the UV spectrum of I in hydrochloric acid solutions (from 1.0 to 10.0 mol dm-3) are similar to those previously published for perchloric and sulfuric acid solutions.11,13 In hydrochloric acid solutions with the concentration higher than 1.0 mol dm-3, a medium effect on the absorption curves causes loss of the isosbestic points. The application of the CVA method, for ten UV spectra of I in hydrochloric acid solutions is resulting in appearance of two isosbestic points at 236 and 292 nm. The first vector accounts for 91.6 % of the variability. It is possible, that 3-C atom is protonated in this case of (See sheme 1 - structure III). Podkovinska et al.16 observed two bands in UV spectrum of the protonated form of I in sulfuric acid at wavelengths at 258 and 262 nm. In this work, in UV spectra of I at concentration of phosphoric acid from 10.5 to 11.5 mol dm-3 and hydrochloric acid solutions 7.5 to 8.5 mol dm -3, bands at wavelengths about 260 nm are not obtained. Similarly, in UV spectra of I in sulfuric acid solution or perchloric acid solutions, bands at A>260 nm in this region are not obtained.11,13 Therefore, it is possible, that the new bands appearing at 258 and 262 nm result from the formation of a new reaction product during the process of protonation (isomerization or oxidation of protonated form of I). B. S. Andonovski, G. M. Stojkovič: UV Study of Indole and 3-Acetylindole in Phosphoric and… H I 356 Acta Chim. Slov. 2000, 47, 349-362. 2. UV spectra of 3-acetylindole in water, in phosphoric acid and hydrochloric acid solutions Figure 3 shows six UV spectra of 3-AI in water and phosphoric acid solutions at concentration from mol dm-3 to mol dm-3. UV spectrum of 3-AI shows in water media absorption maxima at: 206 nm (1Ba), log {e} = 4.38; 242 nm (1Bb), log {e} = 4.02; 260 nm(1La), log {e} = 3.93;, 300 nm (1Lb), log {e} = 4.03. Changes in the UV spectrum of 3-AI (1Ba, 1Bb, 1La and 1Lb bands) in phosphoric acid solutions in the concentration range from 4.5 to 11.0 mol dm-3 are noticeable, of similar for those in perchloric or sulfuric acid solutions.12,14 UV spectra of 3-AI in phosphoric acid solutions are not showing isosbestic points, indicating the solvent influence within the used range of phosphoric acid concentrations. 0.8 0.6 0.4 0.2 'A /A\ ¦ fern I |\| 1 ^ a J \ / VY\ /f\ It^k /