Micelle-catalyzed interaction between glycine and ninhydrin with and without added salts

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1 Indian Journal of Chemistry Vol. B, December 1, pp Micelle-catalyzed interaction between glycine and ninhydrin with and without added salts Kabir-ud-Din*, Jamil K J Salem & Sanjeev Kumar Chemistry Department, Aligarh Muslim University, Aligarh, India and Zaheer Khan Chemistry Department, Jamia Millia Islamia, Jamia Nagar, New Delhi 11 5, India Received October ; accepted (revised) 1 June 1 Reaction of glycine with ninhydrin in sodium acetate-acetic acid buffer solutions (PH = 5.) at C is catalyzed by cationic micelles of cetyltrimethylammonium bromide (CTAB) and cetylpyridinium bromide (CPB). The catalytic efficiencies of these micellar systems increase by some added electrolytes for the nucleophilic attack of amine group on carbonyl carbon of ninhydrin. The catalysis can be related to the extent of incorporation/association of reactants into/with the micelles. The efficiency of anions as catalyst follow the sequence: benzoate > chloride :: bromide :: salicylate > tosylate > sulphate. Cetyltrimethylammonium micelles catalyze the rate of alkaline hydrolysis of benzylpenicillin with a rate enhancement of ca. 5-fold. However, the hydrolysis rate is inhibited by increasing concentrations of hydroxide and penicillin anions I. Recently, similar observations have been reported on some amino acid (glycine, alanine, valine, leucine, isoleucine and phenylalanine)-ninhydrin reactions It was observed that at high concentrations (amino acid.5, ninhydrin.5 mol dm - ) rate inhibition occurred in micellar systems while the rate in bulk aqueous phase increased. We have chosen such reactions to be studied in detail at low reactant concentrations to elucidate better the kinetic micellar effects - 5 In the present work the reaction between glycine and ninhydrin has been studied in solutions containing cationic micelles. In addition, the effect of added salts on the rate were also explored because salts, as additives, in micellar systems acquire a special place due to their ability to induce structural changes 6 which may, in turn, modify the substrate-surfactant interactions. Experimental Section Materials. Ninhydrin, glycine, cetyltrimethylammonium bromide (CTAB) and cetylpyridinium bromide (CPB) were the same as used earlier - 5. All inorganic and organic salts and other chemicals were reagent grade commercial products. Stock solutions of ninhydrin and glycine were prepared in sodium acetate-acetic acid buffe? of ph 5.. The ph measurements were made using ELICO U-lO phmeter. Kinetics. The reaction in presence of CT AB and CPB micelles was studied spectrophotometrically by monitoring the appearance of purple colour (vide infra) as a function of time at 5 nm using a Bausch & Lomb Spectronic spectrophotometer. A threenecked reaction vessel (fitted with double-walled condenser to check evaporation) containing required volume of glycine solution, surfactant and salt (when required) were kept immersed in an oil-bath thermostated at the desired temperature (±. 1 QC). For stirring and to maintain an inert atmosphere, pure nitrogen gas (free from CO and O ) was bubbled through the reaction mixture. The reaction was initiated by adding thermally equilibrated ninhydrin solution; the zero-time was taken at half of the addition of required volume. The experiments were carried out under pseudo-first order conditions by taking [ninhydrin] in excess (at least x). The pseudo-first order rate constants (k'') were calculated upto 8% completion by using k'' = (./t) log (Aoo-AJAoo-At) equation with the help of a computer program (average linear correlation coefficient, r =.989; Aoo = absorbance at infinite time, Ao = absorbance at zero time, and At = absorbance at time t). The critical micelle concentrations (cmc) of CT AB and CPB were determined conductometrically at QC

2 KABIR-UD-DIN et al.: MICELLE-CATALYZED INTERACTION BETWEEN GLYCINE AND NINHYDRIN.. u C CI... III. ct A (nm) Figure I - Spectra of the reaction product of ninhydrin (5. x 1 - mol dm- ) and glycine ( x 1 - mol dm- ) in the presence of 1 x 1 - mol dm- CTAB ( I ), 5 X 1 - mol dm ) ) ) CTAB (), 5 x 1 - mol dm' CPB (), x 1 - mol dm- CPB () and in the absence of surfactant (* ) in buffer (ph=5.) at C. (5) is the spectrum of product obtained by carrying out the reaction at 95 C in absence of surfactant. The spectra were recorded after h of mixing the reactants , has two absorption maxima in the visible range8 ; Amax = 5 and 1 nm. The absorption maximum at 5 nm is usually utilized for qualitative and quantitative studies involving the ninhydrin reaction. No change in the absorption maximum was observed at 5 nm in presence of varying concentrations of CTAB or CPB. These results (Figure 1) show that the purple-co )Ured reaction product is the same In presence of micelles as in aqueous solution. At C (Figure 1), the intensity of colour in aqueous medium is negligible at [glycine] = X 1 mol dm- but, in the presence of surfactant (CTAB or CPB), the reaction is strongly catalyzed and could be performed spectrophotometrical I y. Effect of [surfactanth. To study the effect of [surfactant] on the reaction rate of purple colour formation, the kinetic runs were performed within the [surfactanth from.5 to. 1 mol dm- at fixed [glycine] = x 1 - mol dm, [ninhydrin] = 5. x 1- mol dm at C. The results are shown graphically in Figure as rate constant (kljl) - [surfactant] profiles. Table I contains calculated values of the pseudo-first order rate constants (kljlcal), according to Eqn (9) (vide infra), which shows a close agreement. Effect of [glycine] and [ninhydrin]. In order to confirm the mechanism in micellar medium vis-a-vis aqueous medium, several kinetic runs were carried out at. mol dm- CTAB and various ninhydrin and glycine concentrations. The results are given in Table II. O.O L ( S u r f a c ta n t J ( m o l d m - ). 1 Figure -Effect of [surfactant] on the reaction rate of ninhydrin with glycine (e,, for CTAB and CPB, respectively). Reaction conditions: [ninhydrin] = 5. x 1 - mol dm-, [glycine] = x 1 mol dm-, ph=5.. temp. = C. in presence of glycine( x 1 - mol dm- ) and ninhydrin (5. x 1- mol dm- ). The values are 1. x 1- and 1.6 x 1- mol dm- for CTAB and CPB, respectively, which are lower. than the corresponding values in the aqueous medium: ( 1.7 x 1- and 1.8 x 1- mol dm- ). Results Spectra of the reaction product. Spectra of the product of glycine-ninhydrin reaction (purple colour) Effect of added salts. The effect of addition of inorganic and organic salts on kljl was studied at fixed concentrations of x 1 - mol dm- of glycine, 5. x 1- mol dm- of ninhydrin and. mol dm- of CTAB at C. The results are presented graphically in Figures and. Effect of temperature. A series of kinetic runs was carried out within the temperature range 65-8 C at fixed [ninhydrin] (= 5. x l O- mol dm- ) and [glycine] (= l.o x 1 - mol dm- ) in the presence of. mol dm- CTAB or. mol dm- CPB. The data obtained were found to fit the Eyring Eqn ( 1 ).... (1) where kb' h, S#, R and H# are, respectively, Boltzmann's constant, Planck's constant, activation entropy, the gas constant and activation enthalpy. The activation parameters were calculated using linear

3 INDIAN J CHEM. SEC B. DECEMBER Table I - Values of the calculated rate constants (klilcah using Eqn (9» and agreement between calculated and observed ( kill ) rate constants for the reaction of ninhydrin with glycine shown as a function of [surfactant] at temp. C. ph = 5.. [glycine] x 1 - mol dm' and [ninhydrin] = 5. x 1- mol Table II-Effect of [glycine]. [ninhydrin] and temperature on the pseudo-first-order rate constants (kw) for the reaction of ninhydrin with glycine at ph=5. and [CTAB] =. mol dm- = dm- = 1O\IlCal (S- I ) [surfactant] (mol dm- ) CTAB CPB CTAB CPB Q l CHz- C(X)H I IOs[ninhydrin] Temp. (mol dm'j) (C) (kill kwcal )lkw [glycine] (mol dm' ) I Oskljl (S' I ) l (.9)" (5. 1 ) 7.9 ( ) 1. (9.8) "Values in parentheses pertain to. mol dm'j CPB o + 1,& ~ N-C -COOH i) decarboxylation ii) hydrolysis o N + O o - Q- O N o 5 Scheme I least-squares regression technique. The values of H# (k J mol- I ) and S# (J K- I mor l ) were found. respectively, to be 1 66., -55. (CTAB, r =.998) and 1 58., (CPB, r =.998). Discussion The mechanism of the a-amino acid-ninhydrin reactions in aqueous medium has been detailed on many occasions,5,9, 1O, The reaction (Scheme I) proceeds

4 KABIR-UD-DIN et at.: MICELLE-CATALYZED INTERACTION BETWEEN GLYCINE AND NINHYDRIN concentration range of reactants related to that observed at high reactant concentrations - instead of inhibition the results show catalysis. Situations like this (i.e., inhibition at high reactant concentrations) have been explained when both reactants compete for the same type of sites in the micelle and when both are required to bind to the micelle l. Under such a situation, the reaction of glycine(gly) and ninhydrin(nin) at different surfactant concentrations may easily be explained in terms of micellar pseudo phase model proposed by Menger and Portnoy l and developed by Bunton l and Romsted ' 5 (Scheme II).. K (gly)w + On.. s... (giy)m. L-...:...L L...L.6 L.8 [Salt] (mol d m- ) rate of ninhydrin with glycine at x 1. mol dm- CTAB in buffer (ph=5.) at C. through the formation of a Schiff base. After decarboxylation and hydrolysis of the Schiff base, amino-indandione is formed as stable intermediate which is very reactive. Finally, a second molecule of ninhydrin condenses with producing Ruhemann's pulple S. The reaction product (Ruhemann's purple) has been well characterized which possesses two absorption maxima in the visible ranges. In our present studies in micellar systems, the reaction products carbon dioxide and formaldehyde were detected by known methods 1 1. Evolution of CO and aldehyde as well as dependence of rate of purple colour formation on [glycine] (first order) and [ninhydrin] (fractional order) were found similar to those in the aqueous medium9. These, alongwith the spectral observations (vide supra), demonstrate that the mechanism remains unchanged. Let us now take into account the kinetic data in micellar systems. Figure shows that the pseudo-first order rate constant increases upon increasing the [surfactant] at low surfactant concentrations and reaches a maximum at about.5 mol dm-. Further increase in surfactant concentration eventually leads to a slow decrease. The profile shape is perfectly general being a common characteristic of bimolecular reactions catalyzed by micelles 1-1. We see that the situation is completely reversed III the low... ()... () -l--l 1. Figure -Effect of [NaBr], [NaCl] and NaS] on the reaction (gly)w + (nin)w (giy)m + (nin) m kw km. purple colour -I purpie colour... ()... (5) Scheme II Here On is the micellized surfactant and w and m refer to aqueous and micellar pseudo-phases. Reaction in water (step ) is neglected due to negligible reaction in aqueous medium (almost no purple colour developed at this temperature in the absence of surfactant, Figure 1) at C. As such, the observed first-order rate constant is given by kill = (Ks km [On)) / ( l + KdOn)).. (6). where [On] = [surfactanth - cmc, and km is the first order rate constant in micellar medium and related to the second order rate constant, k'm, as S (7) km = ( k ' m [ ( n i n ) m) ) / [ O n] = k'm M N S where mole fraction, M N, is given by M SN = [ ( n i n ) m] / [ O n] (8) Equation (6) can be written as Eqn (9) when km-value is substituted from Eqn (7)... (9) kill = ( K s k ' m [ O n] M SN)/( l + K s [ On] ) S Values of M N were estimated by considering Eqn () in Scheme II and the mass balance to total ninhydrin concentration 1 6. Using a non-linear regression computer program where KN was an adjustable parameter, the best values of Ks and k'm were obtained which are given in Table III (details of the procedure are given elsewhere-5 ).

5 INDIAN J CHEM, SEC B, DECEMBER 1 1 Table III- Value of KN, Ks and k'm for the reaction of glycine with ninhydrin at ph 5. = 8.. Parameter 1. 8 KN (mol- I dm) Ks (mol- I dm ) 5 1 k'm (S-I) 1.6 C'" [Sa lt] ( m o l d m - ) Figure - Effect of organic salts on the reaction rate of ninhydrin with glycine at x 1 - mol dm- CTAB in buffer (ph=5.) at C. KN was also determined independently by spectral shift method 17 The chosen wavelength was 5 1 nm at which the changes in absorbances were maximum for the ninhydrin binding with CTAB. The [ninhydrin] was. x 1-5 mol dm- and the [surfactant] was varied up to x 1 - mol dm The value of KN was found to be 1 78 mol- t dm which is of the same order as our kinetic value and it seems that using KN = 1 mol- 1 dm under the kinetic conditions is justified. Glycine participates in the following acid-base equilibria' 8 H:! N+ Cl-l:COOH-- Ka1 -- H:! N+ CH:!COO"-- 1 Ko H:! N CI-I: COO H Ka-- H:!NCI-i:!COO" with Ka l =. x 1, Ka = 1.6 x 1-1 and Ko ::. 15 Obviously, the zwitterionic form H N+CHCOO- is the major existing species under our experimental conditions of ph 5.. On the other hand, in its all likelihood it forms an ion-pair between the anionic site (-COO-) and the cationic head groups of CTAB/CPB micelles. Therefore, the electrostatic interactions between the cationic micelles and the carboxylate ions assist in localisation of glycine near to the micelle-water interface. It has been ascertained8 1 that HNCHCOOH form is reactive than H N+CHCOO- towards the nucleophilic attack on the carbonyl group of ninhydrin. The presence of 1t-electrons in ninhydrin 1 9 increases the possibility of its partitioning between the aqueous and positively charged micelles. Therefore, both the reactants may - CTAB CPB get effectively incorporated/associated into the aqueous surface of the micelles (i.e., Stern layer, which is a possible site for the reaction, Figure 5). Thus, concentration of reactive species increase in the micellar reaction zone, catalyzing the reaction and increasing the observed rate (k\jl)' After the incorporation/association of bulk of glycine and ninhydrin into the micelles, addition of more surfactant generates more cationic micelles which simply take up the ninhydrin and glycine into the Stern layer, and thereby deactivate them, because glycine in one micelle may not react with ninhydrin in another. The reaction rate in CTAB is slightly different than in CPB which indicates that delocalization of charge is less in CPB. Glycine is a polar organic compound. The presence of a salt generally decreases the aqueous solubility of organics As we add NaCI or NaBr, initially (at low concentration range) glycine could be driven off towards micellar surface where it interacts with ninhydrin and a rate enhancement takes place (Figure ). At higher NaCI or NaBr content, the general exclusion of the reactants takes place from the reaction site. This factor operates to decrease the reaction rate because reactant concentrations at the reaction site will decrease in the presence of higher [salt]. However, with NaS an initial decrease and then an increase is observed. As SO contains a higher negative charge, its exclusion effect dominates from the beginning and a decrease in k\jl was observed (Figure ). The kinetic results obtained at higher [NaS ] (increase in k\jl) show that more than one factor are involved (solubility, exclusion, hydrophilicity of the added counterion, etc.). The hydrophobic salts such as sodium tosylate (NaTos), sodium salicylate (NaSal) and sodium benzoate (NaBenz) produce rate enhancement at low concentrations, passing through a maximum as the [salt] is increased (Figure ). Addition of organic hydrophobic salts causes negatively charged counterions to get solubilized in micellar palisade layer with acidic groups exposed near to head group region 1. Therefore, in addition to neutralization of

6 KABIR-UD-DIN et al.: MICELLE-CATALYZED INTERACTION BETWEEN GLYCINE AND NINHYDRIN 11 Table IV -Dependence of the observed pseudo-first order rate constants (kill) on the side chain (R) of the amino acid for the reaction. amino acid + ninhydrin R surfactant CT AB=. CPB=. (mol dm' ) (mol dm' ) ----i. Ruhemann's purple Ref Figure 5-Schematic representation showing probable location of the reactants for the CT AB-catalyzed interaction between glycine and ninhydrin. micellar surface charge, they will restrict solubilization sites to reactants. Thus, they catalyze the reaction by virtue of increased concentration of reactants in the Stem layer. The decreased rate observed at higher [organic salt] is a consequence of adsorption of hydrophobic anion at the micellar surface and exclusion of substrate from the micellar surface. The progressive withdrawal of the substrate from the reaction site would slow down the rate as indeed observed. Another factor which could inhibit the rate is the possible. micellar growth at higher [organic salt] as reflected by viscosity data (Figure ). In our case the change in morphology from spheroidal micelles to rod-shape would bring certain changes on the characteristics of the micelle. As the [surfactant] is constant, micellar growth will cause decrease of number density of micelles with a concomitant decrease in catalytic efficiency of the system. In the formation of Ruhemann's purple by the interaction of ninhydrin with a-amino acids, various steps are involved and various intermediates are formed during the course of reaction Out of these, -amino-indandione is the most stable and behaves as a reactant for the formation of Ruhemann's purple (Scheme I). The reaction of with ninhydrin is same for all a-amino acids. Therefore, the rates of colour formation are invariant for each amino acid in aqueous medium where the side chain of amino acid is reflected only in the values of rates of CO and RCHO formations (decarboxylation and RCHO formation are fast and are over within min whereas the intensity of purple colour is negligible during this H II II this work '[amino acid) = LOx 1- mol dm-, [ninhydrin) = 5. x 1- mol d - m. period9). Thus, it has been found that R has no significant effect on the rate of purple colour formation, in aqueous medium. In presence of surfactants (CT AB or CPB), it is observed that side chain of the a-amino acid plays an important role: the reaction rate increases with the hydrophobicity of R; the order being for tryptophan, phenylalanine, leucine and glycine (Table IV). It is not advisable to arrange lysine and aspartic acid5 in this sequence because hydrophobicities of the: side chains of these are diminished due to the presence of different types of groups, i.e. -NH and -COOH, respectively. It seems that decrease in the hydrophobicity (from tryptophan to glycine) decreases the effective concentration of the amino acid at the Stem layer of the micelles. Though the data are not conclusive, the trend certainly shows that the increased hydrophobicity is responsible for higher concentration and hence different rates for different amino acids. Thus, unlike aqueous system results, those in micellar systems

7 1 INDIAN J CHEM, SEC B, DECEMBER I show a differentiating influence on the ninhydrin reaction that depends on the hydrophobicity of amino acid side chain, R. On comparing the M! and!!.s' to those of aqueous system9 (M! =. kj mol- I and!!.s' = -5 J K- I mol- I ), fairly high positive values of enthalpy of activation (M! = and kj mor l for CTAB and CPB, respectively) indicate that the transition state is highly solvated in the micellar system. The magnitudes of!!.s' (=-55. and JK- ' mol- I for CTAB and CPB, respectively) are not significantly affected in presence of surfactants; showing that the same mechanism is being followed in aqueous as well as in micellar media. Finally, we can conclude that molecule-molecule interactions in micellar media could successfully be treated using the pseudo-phase model. Quantitative treatment of the kinetic data seems j ustified as k'l' and k'l'cal are in close agreement within experimental errors. Estimation of Ks values look conceivable as calculated in the real kinetic conditions. The effect of salts on micellar catalysis seems to depend on the nature of salt which could accelerate/inhibit the reaction. References I Gensmantel N P & Page M I, J Chem Soc, Perkin Trans //, 198, 1 7. Khan Z, Ali S I, Rafiquee Md Z A & Kabir-ud-Din, Indian J Chem, 6A, 1997, 579. Kabir-ud-Din, Salem J K J, Kumar S, Rafiquee Md Z A & Khan Z, J Colloid Interface Sci, 1, 1999,.. Kabir-ud-Din, Rafiquee Md Z A, Akram M & Khan Z, Int J Chern Kinet, 1, 1999, Kabir-ud-Din, Salem J K J, Kumar S & Khan Z, J Colloid Interface Sci, 1 5, 1999, 9; Colloids Surf A, 1 68,, 1 ; Indian J Chem, 9A,, Imae T & Ikeda S, Colloid Polym Sci, 65, 1987, 1 9. Hydrogen Ions, Vol I, edited by H T S Britton (Chapman and Hall, London), 19. Friedman M & Sigel C W, Biochemistry, 5, 1966, 78. Khan Z & Khan A A, J Indian Chem Soc, 66, 1989, 5; 67, 199, 96. McCaldin D J, Chem Rev, 6, 196, 9. Feigl F, Spot Tests in Organic Analysis (Elsevier, Amsterdam), 196. Menger F M & Portnoy C E, J Am Chem Soc, 89, 1967, 698. Bunton C A & Robinson L, J Am Chem Soc, 9, 1968, 597; Bunton C A, Catal Rev - Sci Eng,, 1979, I ; Bunton C A & Savelli G, Adv Phys Org Chern,, 1986, 1. Bunton C A, J Mol Liq, 7, 1997, 1. Romsted L S, i n Micellization, Solubilization and Microemulsions, Vol., edited by K L Mittal (Plenum, New York), 1977; Romsted L S, in Surfactants in Solution, Vol., edited by K L Mittal & B Lindman (Plenum, New York), 198. Bunton C A, Gan L-H, Moffatt J R, Romsted L S & Savelli G, J Phys Chern, 85, 1981, ' Bunton C A, Rivera F & Sepulveda L, J Org Chem,, 1978, Friedman M & Wall J S, J Am Chem Soc, 86, 196, 75. JouIlie M M, Thompson T R & Nemeroff N H, Tetrahedron, 7, 199, Hoiland H, Ljosland E & Backlund S, J Colloid Interface Sci, 1 1, 198, 67. Lin Z, Cai J J, Scriven L E & Davis H T, J Phys Chem, 98, 199, 598. Steigman J, Cohen I & Spingola F, J Colloid Sci,, 1965, 7. Lamothe P J & McCormick P G, Anal Chem, 5, 197, Khan Z, Ph D thesis, Aligarh Muslim University, Aligarh, India,