The Micelle-Induced Interaction between Ninhydrin and Tryptophan

Journal of Colloid and Interface Science 215, 9 –15 (1999) Article ID jcis.1999.6211, available online at http://www.idealibrary.com on

The Micelle-Induced Interaction between Ninhydrin and Tryptophan Kabir-ud-Din,* ,1 Jamil K. J. Salem,* Sanjeev Kumar,* and Zaheer Khan† *Department of Chemistry, Aligarh Muslim University, Aligarh-202 002, India; and †Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi, India Received March 26, 1998; accepted March 16, 1999

two cationic surfactants having different head groups, namely, cetyltrimethylammonium bromide (CTAB) and N-cetylpyridinium bromide (CPB). This paper contributes experimental evidence of the catalytic effect of the cationic micelles on the title reaction under the experimental conditions.

The effects of micelles of cetyltrimethylammonium bromide (CTAB) and cetylpyridinium bromide (CPB) on the observed pseudo-first-order rate constants for the interaction of ninhydrin with tryptophan were studied. The influence of different parameters was considered, i.e., reactant concentration, surfactant concentration, temperature, and effect of added salts. The data are explained in terms of the pseudo-phase model of the micelles. © 1999 Academic Press Key Words: micelle; surfactants; micellar catalysis; kinetics; ninhydrin; amino acid; tryptophan; cetyltrimethylammonium bromide; cetylpyridinium bromide.

EXPERIMENTAL

Materials CTAB, CPB, and N were the same as used earlier (7). Trp, NaCl, and NaBr were Merck products (99%) while sodium tosylate (NaToS) was a Fluka product (99%), and all were used as received. An acetate buffer of pH 5, which was used as solvent for preparing all stock solutions, was prepared in doubly distilled and deionized water (specific conductance (1 2 2) 3 10 26 ohm 21 cm 21).

INTRODUCTION

The use of ninhydrin (N) for the detection/estimation of amino acids has great potential in revealing latent finger prints (1). The use depends on the formation of Ruhemann’s purple (2). The method, though useful, still has much room for improvements. Continuous efforts are, therefore, being made to improve the method (1, 3). Micelles are known to provide different microenvironments as there is a nonpolar, hydrophobic interior that can provide binding force for similar functionalities on the reactant and a polar, usually charged, palisade layer that can interact with reactant’s polar parts (4). Due to these facts, a significant amount of systematic kinetic results have been reported on the effect of micelles on various organic reactions during past few decades (5). Several photochemical studies comprising tryptophan (or related indolic compounds) have been carried out in micellar medium in order to mimic some of the characteristics of biological membranes (6). It has recently been observed that micelles catalyze and inhibit the reactions of N with a-amino acids and their metal complexes (7). Rossi et al. (8) have concluded that, at pH 10, tryptophan (Trp) has been incorporated to cationic micelles but remains in the aqueous phase in anionic or nonionic ones. In search of an enhanced utility of ninhydrin–amino acid research we tried reaction of Trp in a micelle mediated aqueous medium under a set of conditions. For this purpose, we used 1

Kinetic Measurements Trp and the surfactant solutions were taken in a three-necked reaction flask fitted with a double-surface condenser to prevent evaporation. The flask was then kept thermostated in an oilbath at the experimental temperature (accuracy 60.1°C). The reaction was started by addition of a thermally equilibrated requisite volume of the ninhydrin solution. Zero-time was considered to be when half of the volume of N had been added. A slow stream of pure N 2 gas (free of CO 2 and O 2) was continuously passed through the reaction mixture for stirring, as well as to maintain an inert atmosphere. Progress of the reaction was followed spectrophotometrically (Bausch and Lomb Spectronic-20) by measuring the absorbance of the reaction product (purple colored) at 570 nm (9). Pseudo-firstorder conditions were adopted by taking N in excess. The observed pseudo-first-order rate constant (k c ) was obtained up to completion of three half-lives by using k c 5 (2.303/ t)log( A ` 2 A 0 )/( A ` 2 A t ) with the help of a computer program. In the present study, the kinetic runs were carried out as a function of [Trp] T, [N] T, [surfactant] T, and temperature (65– 80°C). Critical micellar concentration (cmc) values of CTAB and CPB were determined conductometrically (10) at 70°C in the

To whom correspondence should be addressed.

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0021-9797/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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KABIR-UD-DIN ET AL.

presence of the Trp and N that were used to obtain the concentration of the micellized surfactant ([D n ]) (vide supra). To test possible micellar growth in our systems, especially in the presence of salts, solution viscosities ( h r ) were determined by using a modified Ubbelohde viscometer (11). RESULTS AND DISCUSSION

Before disembarking on the kinetic results of the ninhydrin– tryptophan reaction in micellar media and pertaining interpretations/explanations, it seems appropriate to consider the mechanism of the ninhydrin reaction in general. The mechanism (Scheme 1) involves, as the first (and rate-determining) step, formation of a Schiff base (B), which is a case of the generalized condensation of a carbonyl compound with an amine (12). The intermediate C is then produced by electronic changes which involve decarboxylation and dehydration. The decarboxylation step is not the rate-determining because at pH 5 it is expected to be unimolecular and not subjected to steric hindrance. The hydrolysis of C to 2-aminoindanedione (D 1) could not be rate controlling either because the rates should be governed by steric factors alone (13). The reaction between D 1 and ninhydrin also involves a nucleophilic type of displacement of a hydroxy group of ninhydrin, leading to the formation of diketohydrindylidenediketohydrindamine (DYDA, also called the Ruhemann’s purple). (Formations of CO 2 and aldehyde were confirmed by following the same procedure described earlier (14)). The intermediate D 1 participates in two parallel reactions (Scheme 1). At pH , 5.0, the reaction proceeds chiefly by route (b), where ammonia is evolved quantitatively and no purple color is formed. In solutions of pH $ 5.0, route (a) predominates and, under these conditions, color formation is the basis of the analytical method (9). Therefore, to obtain the highest color yield, all the studies were performed at pH 5.0. Under our experimental conditions, the existence of the anionic form of tryptophan (RCH(NH 2)COO 2) is negligible because the pK 2 of tryptophan is 9.39 (12). The cationic (RCH(N 1H 3)COOH) and zwitterionic (RCH(N 1H 3)COO 2) species have positive charges on the nitrogen atom of the amino group and could not participate in a nucleophilic displacement reaction (as the unshared electron pair of nitrogen, responsible for attack on the carbonyl carbon of ninhydrin, is combined with a proton) (14, 15). The neutral species (RCH(NH 2)COOH), which exists in equilibrium with the zwitterionic species (16), is therefore expected to react with ninhydrin. No doubt, the zwitterionic form will cause the substrate molecule to come closer to the micellar head group region (due to ion-pair formation between the anionic carboxylate site and the cationic head groups). The concentration of tryptophan thus increases within the outer aqueous areas of the micelles. The electrostatic interactions between the cationic micelles and the

2COO 2, therefore, assist in localization of the tryptophan near the micelle–water interface. On the other hand, the presence of p-electrons in ninhydrin increases the possibility of partitioning between water and positively charged micelles. Therefore, the overall increase (catalysis) of the reaction rate is due to concentrating both the reactants in the micellar head group region (Stern layer). Figure 1 shows that at 70°C with [Trp] T 5 1.0 3 10 24 mol dm 23 and [N] T 5 5 3 10 23 mol dm 23 no reaction occurs in an aqueous medium, and therefore, the observed absorbance was almost negligible (Figure 1, curve marked with asterisks). In contrast, under the same conditions in the presence of CTAB or CPB micelles, the purple-colored product was formed (l max 5 570 nm). When the temperature of the reaction was raised to 95°C, the same product was formed in an aqueous medium also without micelles with the same l max (5570 nm, Figure 1, Curve 5). This confirms that the product of the reaction, diketohydrindylidenediketohydrindamine (DYDA) (1, 9), thus remains unchanged with the change in the reaction medium from aqueous to micellar pseudo-phase. Dependence of k c on [Reactants] Table 1 summarizes the pseudo-first-order rate constant’s (k c ) dependence on reactant concentrations. The k c was found to be independent on the initial concentration of Trp in the range (0.8 –3.0) 3 10 24 mol dm 23, whereas with an increase in [N] T from 0.005 to 0.030 mol dm 23, the k c increased by nearly threefold (in both cases the [CTAB] T was fixed at 0.02 mol dm 23). We thus see that the reaction order in the micellar medium is first and fractional (Fig. 2), respectively, with respect to the [Trp] T and [ninhydrin] T. These results and observations of product formations (e.g., CO 2, RCHO, and DYDA), in comparison with those of several amino acid–ninhydrin reactions in an aqueous medium (1, 7, 9, 14), lead us to safely conclude that the mechanism in the presence of cationic micelles remains the same as that in the homogeneous aqueous medium with all possible intermediary situations. Dependence of k c on [Surfactant] The k c values listed in Table 2 show a nearly fourfold increase with an increase in [surfactant] T from 0.005 to 0.070 mol dm 23 with fixed [Trp] T and [N] T at 70°C. The catalysis observed under the present experimental conditions (Fig. 3) is in agreement with similar bimolecular reactions (17), and the catalytic effect of CTAB or CPB on k c may be explained in terms of the pseudo-phase model of micelles (18) (Scheme 2). The observed k c is given by Eq. [1], as the reaction in water (k w) is neglected because the purple color does not appear at 70°C (Fig. 1). kc 5

K Sk m@Dn # 1 1 K S@Dn #

[1]

INTERACTION BETWEEN NINHYDRIN AND TRYPTOPHAN

SCHEME 1

11

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KABIR-UD-DIN ET AL.

FIG. 1. Spectra of the reaction product of ninhydrin (5.0 3 10 23 mol dm 23) with tryptophan (1.0 3 10 24 mol dm 23) in the presence of 5 3 10 23 mol dm 23 CTAB (1), 30 3 10 23 mol dm 23 CTAB (2), 10 3 10 23 mol dm 23 CPB (3), 60 3 10 23 mol dm 23 CPB (4), and in the absence of surfactant (*) in buffer (pH 5.0) at 70°C. Curve (5) is the spectrum of the product obtained by carrying out the reaction at 95°C in absence of surfactant.

Here, k m and K S are, respectively, the first-order rate constant in the micelles and the tryptophan binding constant to the micelles, and [D n ] is the concentration of the micellized surfactant (5[surfactant] T 2 cmc). Equation [1] can be rearranged to give Eq. [2].

TABLE 1 Effect of [Trp] and [Ninhydrin] on the Pseudo-First-Order Rate Constants (k c) for the Reaction of Trp with Ninhydrin at pH 5.0, [CTAB] 5 20 3 10 23 mol cm 23 and temperature 5 70°C 10 4[Trp] T (mol dm 23) 0.8 1.0 1.5 2.0 2.5 3.0 1.0

10 2[ninhydrin] T (mol dm 23) 0.5

0.5 1.0 1.5 2.0 2.5 3.0

FIG. 2. Effect of [ninhydrin] on the reaction rate of ninhydrin with tryptophan. Reaction conditions, [CTAB] T 5 20 3 10 23 mol dm 23, [tryptophan] T 5 1.0 3 10 24 mol dm 23, pH 5.0, temperature 5 70°C.

1 1 1 1 5 1 z kc km k mK S @Dn #

Equation [2] is useful in that it predicts the linearity of the plots of 1/k c against 1/[D n ] (18, 19) and, as such, the determination of both k m and K S. From the slope and intercept of the linear plots of 1/k c vs 1/[D n ], k m and K S were calculated (r 5 0.991 and 0.976 for CTAB and CPB, respectively) using cmc values from Table 3. The second-order rate constant (k9m) can be written in terms of the first-order rate constant (k m) and the mole ratio of bound N to the micellar head group, M NS (5[N m]/[D n ]), as

10 5 k c (s 21) 20.5 21.4 22.1 21.9 22.5 21.8 21.3 38.3 45.5 57.5 59.4 61.1

[2]

k9m 5

km M SN

[3]

[N m] was estimated from equilibrium distribution constant, Eq. [4], and the mass balance, Eq. [5].

KN 5

@Nm# @NW#~@Dn # 2 @Nm#!

@N# T 5 @NW# 1 @Nm#

[4] [5]

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INTERACTION BETWEEN NINHYDRIN AND TRYPTOPHAN

TABLE 2 The Pseudo-First-Order Rate Constants (k c) for the Reaction of Ninhydrin with Tryptophan in CTAB and CPB Micellar Systems at pH 5 5.0 and Temperature 5 70°C 10 5 k c (s 21)

a b

10 5 k ccal a (s 21)

(k c2 k ccal)/k c

[Surfactant] (mol dm 23)

CTAB

CPB

CTAB

CPB

CTAB

CPB

0.005 0.010 0.015 0.020 0.025 0.030 0.040 0.050 0.070

8.5 b 14.0 17.3 21.4 22.5 23.7 27.5 31.2 33.4

8.0 b 14.3 16.4 19.8 20.6 21.7 24.8 28.1 30.1

13.6 17.8 21.0 23.3 25.1 27.8 29.6 30.0

13.8 17.4 19.9 21.7 23.1 25.0 26.3 27.3

20.02 20.02 10.01 20.03 20.05 20.01 10.05 10.09

10.03 20.06 0.00 20.05 20.06 0.00 10.06 10.09

Calculated from Eq. [1] using k m and K S values given in Table 3. Excluded from the least-squares calculation.

Upon solving Eqs. [4] and [5], quadratic Eq. [6] results, K N@Nm# 2 2 ~1 1 K N@Dn # 1 K N@N# T!@Nm# 1 K N@Dn #@N# T50,

[6]

which was solved for [N m] using the best value of K N 5 100 mol 21dm 3 and [N] T as the total concentration of ninhydrin (5.0 3 10 23 mol dm 23). Estimated values of k9m and K S are given in Table 3. To confirm the validity of rate Eq. [2], the

FIG. 3. Effect of [surfactant] on the reaction rate of ninhydrin with tryptophan (F, E, for CTAB and CPB, respectively). Reaction conditions, [ninhydrin] T 5 5.0 3 10 23 mol dm 23, [tryptophan] T 5 1.0 3 10 24 mol dm 23, pH 5.0, temperature 5 70°C.

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KABIR-UD-DIN ET AL.

there would be delocalization of charge as well as less charge shielding in comparison to in CTAB. In addition to the above fact, there may be an orientational effect, so that the reactive group on the amino acid is less accessible in CPB micelles. These factors undoubtedly affect the interaction with reactant molecules, and consequently, the rates of overall reactions are different. Therefore, higher K S and lower k9m values are expected in the presence of CPB micelles. The values in Table 3 prove these points unambiguously. Effect of Temperature on k c SCHEME 2

rate constants were calculated by substituting the values of k m and K S in Eq. [2] and comparing with k c values. The close agreement between the observed and calculated values (Table 2) supports the validity of the proposed mechanism. The binding constants, K S, for the association of substrates with micelles have been approximated from measurements of the extent to which various concentrations of surfactant increase or decrease the rate of passage of a substrate through columns of a molecular sieve. The differences in the counterion interactions with the micelle and the micelle–substrate complex and activity coefficient effects may seriously complicate the results. The reported K S values for Trp in cationic micelles show a marked dependence upon the pH and the species of Trp which exist at different pH values (6). The K S values of Trp in CTAB, under the present conditions of kinetics, i.e., [ninhydrin] 5 5 3 10 23 mol dm 23 and sodium acetateacetic acid buffer (pH 5.0), were found to be 59 and 80 mol 21 dm 3 for CTAB and CPB, respectively. If one tries to explain these kinetically derived K S values with a K S value equal to that obtained under other conditions, the kinetic data cannot be fitted. Actually, the reaction has been studied in the presence of buffer and ninhydrin, which will have their own effects on the substrate association with the micelles. Therefore, different contributary factors will influence the K S values which could not agree with the K S of Ref (6), where measurements were made at 20°C in aqueous medium at different pH values (adjusted by addition of HCl or NaOH). Though the values differ significantly, the kinetically determined K S values are pertinent for the reason that they belong to the actual experimental conditions. As compared to in CTAB micelles, the second-order rate constant (k9m) is slightly decreased and K S is increased in CPB micelles. This may be due to the fact that the head group size of the surfactant is one of the factors that decides the packing of the surfactant monomers into a micelle; if so, we would expect a difference of packing in the two cases of CTAB and CPB. Of course, with an aromatic pyridinium ring in CPB,

The k c were obtained by conducting kinetic experiments within the temperature range 65– 80°C in the presence of CTAB (0.02 mol dm 23) or CPB (0.02 mol dm 23). Activation parameters such as enthalpy of activation (DH*) and entropy of activation (DS*), obtained from the Eyring equation, are summarized in Table 3. Comparing the values with those obtained in aqueous medium with different amino acids (14), we find that the presence of cationic micelles lowers the DH* with a substantial negative DS*. This lowering occurs not only through the adsorption of both the reactants on the micellar surface but also through stabilization of the transition state. The fitting of the observed k c at different temperatures to the equation was examined (Table 3), and it was found that the Erying equation is applicable to the micellar media, and the sensitivity of micelle structure to temperature is kinetically unimportant.

TABLE 3 The k c Values Obtained at Different Temperatures, Activation Parameters, and Kinetic Results (at 70°C) for the Reaction of Ninhydrin with Tryptophan in CTAB and CPB Micelles 10 5 k c /s 21

Surfactant CTAB a

CPB a

T (°C)

CTAB b

CPB b

12.3 (13.7) 59

10.6 (10.8) 80

65 70

10 4 k m (s 21)

4.0

3.3

75

10 3 k9m (s 21)

5.5

4.6

80

18.0 (17.8) 21.4 (21.7) 28.7 (26.7) 32.7 (31.9)

17.0 (15.9) 19.8 (19.7) 27.5 (24.7) 31.6 (29.4)

10 4 k 2m (mol 21 dm 3 s 21) DH* (kJ mol 21) DS* (JK 21 mol 21)

7.7 c

6.4 c 35.7 2212

37.7 2207

Parameters 10 4cmc (mol dm 23) K S (mol 21 dm 3)

a

The cmc values in parentheses pertain to absence of reactants Trp and N. Values in parentheses are calculated from the Eyring equation; k c 5 (k BT/h)exp[2(DH* 2 TDS*)/RT] using given DH* and DS* values. c Here k 2m is the second-order rate constant in the Stern layer (20) estimated as k 2m 5 0.14 k9m. b

INTERACTION BETWEEN NINHYDRIN AND TRYPTOPHAN

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Once this site is fully occupied, additional [NaTos] will try to get adsorbed at the micellar surface (a case of adsorption) and will thus compete for a site with reactants (a case of tosylate ion association in the form of adsorption). Consequently, [reactants] is decreased at the reaction site by the latter effect (exclusion of substrate). The progressive withdrawal of the substrate from the reaction site would slow down the rate, as was indeed observed. Possibly the above two effects work together on NaTos addition and the resultant effect is a function of [NaTos]. ACKNOWLEDGMENTS The authors thank the Chairman, Department of Chemistry, for facilities and Al-Azhar University authorities for sanction a leave for J.K.J.S.

REFERENCES

FIG. 4. Effect of [salt] on the reaction rate of ninhydrin with tryptophan at 20 3 10 23 mol dm 23 CTAB in buffer (pH 5.0) at 70°C. The effect of [NaTos] on solution viscosity is also shown, which implies that no micellar growth is taking place in the system.

Salt Effect Results Figure 4 shows that the rates decrease with increasing [NaBr] or [NaCl] at 0.02 mol dm 23 CTAB. The inhibitory effect is due to the exclusion of reactants from the reaction site (i.e., Stern layer, as most of the ionic micelle mediated organic reactions are believed to occur in this region) (5). On the other hand, the hydrophobic salt, sodium tosylate (NaTos), gives marked rate enhancement at low [NaTos], passing through a maximum as the [salt] is increased. With such hydrophobic salts, penetration of the benzene ring into the micellar palisade layer (a few carbon atoms deep toward core) takes place with the sulfonate group remaining in the outermost region of the micelle (a case of intercalation) (21). Therefore, in addition to neutralization of micellar surface charge, they restrict interior solubilization of reactants causing an increase in concentration of the latter in the Stern layer; the reaction is thus catalyzed. As we increase the [NaTos], the above site will first be saturated.

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