Kinetics and Mechanism of Ninhydrin Reaction with Copper(I1) Complexes of Glycine and a-Alanine.

Elucidation of the Template Mechanism

DILEEP GUPTA, W E E R KHAN, and A. AZIZ KHAN Department of Chemistry, Aligarh Muslim University, Aligarh-202002, U.P., India

Abstract

Ninhydrin has been found to react with Cu(glycine)+ and Cu(alanine)+ in the ratio of 1:l . The kinetic studies of the reaction were carried out at different concentrations of the reactants at 80°C (pH = 5.0). The reaction proceeds through the formation of a ternary labile complex of ninhydrin with Cu(I1) complexes of glycine and alanine. The kinetics were found to follow pseudo-first-order reaction:.path with respect to Cu(I1)-complex in presence of excess [Ninhydrin]. Formation of a ternary labile complex indicates a template reaction mechanism based on the reactions with coordinated ligands. The variation of pseudo-first-order rate constants with [ninhydrin] was found to be in good agreement with equation

B1 + B2 = [Ninhydrin]

where B1 and B2 are the unknown empirical parameters. The [acetate ion1 has no significant effect on the rate constants. On the basis of observed data a probable mechanism has been proposed. 0 1993 John Wiley & Sons, Inc.

Introduction

The reaction of ninhydrin with amino acids, yields a purple colored product, diketohydrindylidene-diketohydrindamine (DYDA) [ 1,2]. A number of modifications [3-773 have been introduced to increase the stability of the color. The effect of metal ions has been studied with this point of view by several workers [8-11]. Ganpathy [12] et al. developed a copper(I1)- ninhydrin reagent to distinguish between small peptides, a-amino acid amides, and free amino acids by paper chromatopgraphy. Seiji [131 et al. studied the effects of metal complexing agents and ninhydrin on the chromatographic determination of amino acids and related compounds. Although the mechanism of the interaction of ninhydrin with various amino acids has been proposed [14-181 the studies on the kinetics and mechanism of the reaction of ninhydrin with transition metal complexes of amino acids have not been reported. The kinetic studies related with such interactions a m reported in this article.

Materials and Methods

Glycine, a-alanine, ninhydrin (all B.D.H), and copper nitrate (A.R.) were used as such. Solutions of amino acids, ninhydrin, and copper nitrate were

International Journal of Chemical Kinetics, Vol. 25, 437-443 (1993) 0 1993 John Wiley & Sons, Inc. CCC 0538-8066/93/060437-07

438 GUPTA, KHAN, AND KHAN

prepared in sodium acetate-acetic acid buffer solutions. pH measurements were made using Elico LI-10 pH meter. All other reagents used were of reagent grade.

Kinetic Run

The requisite volume of solutions of amino acids, and copper nitrate except ninhydrin were placed in a three-necked reaction vessel fitted with a double surface condenser to prevent evaporation. The reaction was started by adding the required volume of ninhydrin solutions and zero time was taken when half of the ninhydrin solution has been added. The reaction vessel was kept in an oil bath controlled at the desired temperature with in 2O.l"C. The ionic strength was maintained with KNOB solution. Pure nitrogen gas was bubbled through the reaction mixture for stirring as well as to maintain an inert atmosphere. The progress of reaction was followed spectrophotometrically by pipetting aliquotes at different time intervals and measuring the absorbance at 375 nm using a Elico degital spectrophotometer model CL-27. Pseudo- first-order conditions were maintained in all runs by using a large excess of [ninhydrin]. The first-order rate constants were calculated by using the computer Vax- 11.

Composition of the product formed by the interaction of ninhydrin and copper(I1)-amino acid complex was determined by Job's method of continu- ous variations by heating the reactants at 90" for one h and measuring their absorbance at 375 nm.

Results

In our previous studies it was observed that diketohydrindylidene- diketohydrindamine (DYDA), a purple colored compound which is formed by the interaction of amino acid with ninhydrin, shows two major nonmatching peaks in the visible region (A,,, = 400 nm and 575 nm). Copper(I1) reacts with DYDA to give a red colored complex instantaneously at room temperature (30°C). The A,,, for the red colored complex is 510 nm and the structure of this complex has been proposed by Wieland [191. When ninhydrin solution was allowed to react with a solution containing Cu(I1)- amino acid complex at pH 5, only one peak (A,,, = 375 nm) was observed. The spectra of these complexes are given in Figure 1. The composition of the product formed, as determined by Job's method, was found to be 1 : l (ninhydrin: copper(I1)-amino acid complex).

The effect of pH was studied in sodium acetate-acetic acid buffer, over the range of 3.8 to 5.0 at fixed [ninhydrinl = 1.5 X lo-* mol dm-3, [copper(II)- amino acid] = 1.0 X mol dm-3 and temperature 80°C. At pH 3.8 to 4.0 color is not formed. As the pH is increased from 4.0 to 4.5, color develops but is not stable and optimum pH for stable color formation is 5.0, beyond which no measurable reaction is observed due to the formation of basic copper(I1)-nitrate [Cu(N03)2.3Cu(OH)21.

The effect of [amino acid] was studied at 80°C keeping the other pa- rameters constant. As the concentration of these amino acid is increased in comparison to copper(I1) concentration, the formation of purple color is also observed along with the formation of yellow colored complex (A,,, =

KINETICS AND MECHANISM OF NINHYDRIN REACTION 439

325 350 COO 4 5 0 500 550 600 6 5 0 700

WAVE LENGTH - Figure 1. Absorbance spectra of diketohydrindylindene-diketohydrindamine (DYDA): -0. Absorbance spectra of product of Dyda with copper(I1): 0-0. Absorbance spectra of product obtained by the reaction of copper-amino acid complex with ninhydrin: A-A.

375 nm). Due to the formation of purple color it was not possible to study the effect of glycine and a-alanine concentrations quantitatively.

To study the effect of ninhydrin at fmed concentrations of other reactants the kinetic measurements were made with concentrations of ninhydrin ranging from 5.0 x lop3 mol dmp3 to 3.0 x lop2 mol dmP3 at desired temperature. These results are given in Table I. From these results it has been found that the rate constant vary according to eq. (1)

(1) + B2 B1

l’kobs = [Ninhydrin] where kobs is the observed rate constant for the formation of yellow colored complex and B 1 and B2 are the arbitrary constants. The effect of temperature was studied in the temperature range 60” to 95°C. The observed data were found to fit well in the Arrhenius and Eyring equations. The various activation parameters were calculated and the values are given in Table 11.

The effect of acetate ion was also studied at pH 5.0 because it has tendency to form the complex with copper(I1). It was found that acetate ions had no significant effect on the rate constants.

Discussion

In the absence of metal ions, schiff base formation from any carbonyl compound and amine is a two step reaction. Both steps depend upon the pH of the reaction medium. The amine is nuclophilic owing to the unshared pair of electrons on nitrogen. As the acidity of the reaction medium increases, the amine is protonated and becomes nonnucleophilic and the addition on the carbonyl group no longer takes place. Dehydration is also an acid catalyzed reaction, and an increase in acidity (lower pH) should result in an increase in the rate of dehydration. Thus, the reaction is a balance between two steps,

440 GUPTA, KHAN, AND KHAN

TABLE I. = 1.0 X dm-3, H' = 1.0 X

Effect of ninhydrin concentration on the rate constant (kobs); [Cu(II)-glycinel' mol dm-3, ionic strength = 1.0 mol rnol dm-3, [Cu(II)-a-alaninel' = 1.0 X

mol dm-3, and temperature = 353 K.

kobs x lo3 (S-') kobs x lo4 (S-l) [Ninhydrinl X lo2 mol dm-3 (for [Cu(II)-glycine]+) (for [Cu(II)-a-alanine]+)

0.5 0.714 t 0.0030 2.065 t 0.0005 1.0 0.9338 -t 0.0017 4.498 2 0.0010 1.5 1.258 t 0.0023 4.943 2 0.0015 2.0 1.594 t 0.0049 6.230 2 0.0016 2.5 1.715 t 0.0075 6.490 t 0.0020 3.0 1.709 2 0.0010 6.521 t 0.0019

which depend upon the pH in opposite ways. In the presence of metal ions, the formation of schiff base is a one step process [201.

The optimum pH for the reaction of amino acids with ninhydrin has been shown by various workers [15,211 to be in the vicinity of pH 5.0. In these studies, therefore, the pH was varied from 3.8 to 5.0. the reaction was carried with a little excess of [Cu(II)l as compared to [amino acid] to check the side reactions of amino acids with ninhydrin to give the purple color. At pH > 5.0 turbidity due to the precipitation of Cu(I1) was observed and, therefore, the studies were limited up to pH 5.0. In the pH < 5.0 the hydrolysis of schiff base is very fast and decompose simultaneously. Moreover, the metal ions enhance the rate of hydrolysis of schiff base complexes. Therefore, the studies were not carried out at pH < 5.0. The studies with different concentrations of acetate buffer were carried out only to confirm that the complex formation of Cu(I1) with acetate ions is not taking place in the presence of amino acids and is not affecting the kinetics of this reaction.

Pearlmutter and John Stuchr [221 reported the concentrations of various species present in the aqueous solution of glycine and copper(I1) system and observed that the concentration of Cu(g1ycine)n is negligible in comparison to the concentration of Cu(glycine)+ at pH 5.0. Similar results were reported by Negypal et al. [23] who also confirmed that Cu(g1ycine)' is a major existing species in the vicinity of pH 5.0.

Since the studies were carried out at pH 5.0, amino acids exist in the form of zwitter ions. The following ionization equilibria is to be considered.

f

K o 2 NH3- CH- COO-* NH2- CH- COO- + H'

I I R R

TABLE 11. Specific rate constants and activation parameters. [Cu(II)-glycine]' = 1.0 x mol drnv3, [Ninhydrinl = 1.5 X mol dm-3, [Cu(II)-a-alaninel' = 1.0 X rnol

d ~ n - ~ , [H+l = 1.0 X mol dm-3, ionic strength = 1.0 rnol dm-3, and temperature =

353 K.

E: A H + A AS* Complex kobs x lo3 ( s - I ) kJ mol-' kJ mo1-I 5-I J K - ~ mo1-l

[Cu(II)-Glycinel+ 1.258 -t 0.0023 39.18 36.25 7.95 X lo2 -82.81 [Cu(II)-a-alaninel+ 4.943 -t 0.0015 32.16 29.23 2.84 X 10' -94.82

KINETICS AND MECHANISM OF NINHYDRIN REACTION 44 1

The reaction of a-amino acids with ninhydrin is an example of nucleophilic addition reaction. In these reactions lone pair of amino-nitrogen is necessary for the nucleophilic attack on the carbonyl carbon which gives the schiff base (imine). In the case of copper(I1) complexes of glycine and a-alanine, the lone pair of amino-nitrogen is not free due to the coordination to the copper(I1). Therefore, the nucleophilic addition reaction of the complexes with ninhydrin is not possible. When ninhydrin is added to a solution containing [Cu(II) (amino acid)'] complex, formation of a yellow color (A,,, = 375 nm) is observed which probably arises from the formation of schiff base complex [24,25]. The reaction proceeds through the formation of a ternary labile complex of ninhydrin-Cu(I1) (amino acid)+ which is a feature of the template reaction mechanism. In these types of reactions both the reactants must be coordinated with the same metal ion. In Cu(I1) (amino acid12 species the reaction of the complex with ninhydrin is not possible as all the four coordination sites of copper(I1) are fully occupied. In such a case Cu(I1) (amino acid)+ is the only species which may form a ternary labile complex with ninhydrin. Under our experimental conditions (pH = 5.0) unoccupied coordinated positions of copper(I1) could be filled by water molecules. On the basis of the above discussion, and the observed rate data, the following mechanism (Scheme I) has been proposed. The

R R + I Ko2 I

N H + H - C O ~ GNH~- CH - c o 6 t H+ A B

z N

L J

c

+

Scheme I

442

5000 I GUPTA, KHAN, AND KHAN

Y) 0

Y

4000

3000

2 000

1000

0 25 50 1 5 100 125 1 5 0 1'15 200

1 /,CNlNWYDRlNl moi' dm3,

Figure 2. dm-3 and [H'] = 1.0 X [Cu(II) - LY alanine]+ = 1.0 X

Linear dependence of I/kob, against l/[ninhydrinl a t 353 K, p = 1.0 mol mol dm-3; rnol dm-3; [Cu(II) - glycine]+ = 1.0 X

mol dm-3. 0 = glycine; and = a-alanine.

mechanism is in close agreement with the result of various authors on the template reaction mechanism [26-28].

From the mechanism the following rate equation was derived.

or

(3)

(4)

- - d[P] kl K,[C],[Ninhydrin] dt 1 + K,[Ninhydrin]

-

k l K,[Ninhydrin] kobs = 1 + K,[Ninhydrin]

+ B2 - B1

l / k o b s - [Ninhydrin] where B1 = l / k l K , and B2 = l / k , .

Equation (3) on rearrangement gives eq. (41, which shows a linear depen- dence of Ilkobs on l/[Ninhydrin] (Fig. 2) . B I and B2, which are the gradients and intercepts of the plots in Figure 2, were calculated and are given in Table 111.

From the values of B1 and B Z , the values of Kl and kl have been calculated and found to be 66.66, 2.5 X s-l for (Cu(II)-glycine)+ and 46.51, 1.25 X lop3 sP1 for (Cu(I1)-a-alanine)', respectively. From these values it

TABLE 111. Linear parameters corresponding to I / k o b s = Bl/[Ninhydrinl+ B2

Complex

Cu(g1ycine) +

Cu(alanine)+ 6.00

17.20 400 800

Temperature = 353 K, [H'I = 1.0 X mol dm-3, and ionic strength = 1.0 mol dm-3.

KINETICS AND MECHANISM OF NINHYDRIN REACTION 443

is clear that formation of a ternary labile complex is very fast in comparison to the reaction of coordinated amino group to coordinated carbonyl group of ninhydrin. Therefore, kl is the rate determining step and confirms the proposed mechanism.

Acknowledgment

The authors are thankful to Prof. M. S. Ahmad and Prof. Asif-Zaman of this department for mechanistic discussion and also to C.S.I.R., Delhi, for financial support to Zaheer Khan and Dileep Gupta. Thanks are also due to Professor M.A. Beg, Chairman, Department of Chemistry, for providing necessary facilities.

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Received November 1, 1991 Accepted December 15, 1992