Spectrophotometric Investigation on the Kinetics of Decomposition of Murexide in Acid Solutions

Home , Murexide

leohiston noch genügend Bindungszentren vorhan- A n m. b. d. K o r r. : In einer während der Drucklegung die- den sind, um andere, offenbar auch leichter wieder der Arbeit erschienenen Publikation (Biochim. biophysica Acta [Amsterdam] 21, 568 [1956] kommen DAVISON und abtrennbare Proteine zu binden. CHARGAFF und sein BUTLER U. a. auf Grund des konstanten N/P-Verhältnisses 2 12 Arbeitskreis ' haben bei der Fraktionierung von von 4,0 — 4,1 ebenfalls zum Schluß, daß im Thymusnucleo- DN-Histonen mit NaCl-Lösung ansteigender Kon- protein ein wohldefiniertes Molekül vorliegt und nicht eine zentration DNS-Fraktionen von unterschiedlicher lose Histon/Nucleinsäure-Assoziation. Unter Einbeziehung Zusammensetzung und unterschiedlichem Bindungs- des Histidin-Anteils der basischen Aminosäurekomponen- ten des Histons errechnen die Verfasser ein Bindungsver- vermögen erhalten. Die von uns gewonnenen Beob- mögen des Histons von etwa 85% bezogen auf die im Mole- achtungen führen zu dem gleichen Ergebnis. kül vorhandenen Phosphatgruppen, während ohne Histi- din nur etwa 77% zur Bindung kämen. Diesem Prozent- Herrn Direktor G. A. KAUSCHE danke ich für sein satz entspricht ein Molverhältnis von einem basischen förderndes Interesse an dieser Arbeit, Frl. S. RICHTER für ihre technische Hilfe. N-Atom zu 1,3 P-Atomen.

12 C. F. CRAMPTON, R. LIPSHITZ u. E. CHARGAFF, J. biol. Che- LIPSHITZ u. E. CHARGAFF, Biochim. biophysica Acta [Am- mistry 211, 125 [1954]; P. SPITNIK, R. LIPSHITZ u. E. sterdam] 19, 256 [1956]. Vgl. dazu auch G.L.BROWN U. CHARGAFF, J. biol. Chemistry 215, 765 [1955] sowie R. M. WATSON, Nature [London] 172, 339 [1953].

By N. A. RAMAIAH, S. L. GUPTA and J. VISHNU

Department of Physical Chemistry Indian Institute of Sugar Technology, Kanpur (India) (Z. Naturforschg. 12 b, 189—195 [1957] ; eingegangen am 6. November 1956)

The kinetics of the decomposition of murexide in acid solutions were investigated spectrophoto- metrically by following the absorption at ^ = 530 m/<. The optical density of murexide at X = 530 mtt, which obeyed Beer's law, decreased irreversibly with time, following a first order law. The corresponding rate constant k, was dependent upon the concentration (C) of the strong acids like HCl, HBr, HN03, etc. and followed a relationship: Zc/min_1 = 2.56-10~3-C. k was also dependent upon the concentration of murexide. The effect of neutral salts on k showed that in accord with the data obtained with different acids, the anions of the acids or salts had no significant role in the mechanism responsible for the decomposition of murexide. A hypothesis referring to the formation of an intermediate complex (electrically uncharged in character) which decomposed unimolecularly to give the products, uramil and alloxan, was put forward.

1. Introduction teristic absorption spectrum of murexide, for the determination of the amount of Ca++ in sugar cane SCHWARZENBACH and GYSLING 2 described in de- juices, a few interesting results on the decomposi- tail the use of murexide or the ammonium salt of purpuric acid for estimation of a number of metal ions tion of murexide in acid solutions have been re- especially Ca++. Since then, murexide has been ex- corded. It is known that murexide undergoes de- tensively employed for quantitative estimation of composition in acidic solutions to give primarily 6 7 calcium content in pharmaceutical products3, milk uramil and alloxan ' . No data exist in the litera- ultrafiltrate4, etc.5. Estimation with ease of Ca++ ture on the kinetics of this reaction. The present in sugar cane juices is a problem in sugar tech- communication reports a resume of the work done nological laboratories. During a series of trials for in this laboratory on the kinetics of the decomposi- the development of a method employing the charac- tion of murexide.

1 G. SCHWARZENBACH u. H. GYSLING, Helv. chim. Acta 32, 1314 4 H. FLASCHKA, Textil-Rundschau [St. Gallen] 9, 77 [1954]. [1949]. 5 W. TH. G. M. SMEETS, Nature [London] 169, 802 [1952]. 2 G. SCHWARZENBACH U. H. GYSLING, Helv. chim. Acta 32, 1484 B D. DAVIDSON U. E. EPSTEIN, J. org. Chemistry 1. 305 [1936]. [1949], 7 I. HEIBRON U. H. M. BUNBURY, „Dictionary of Organic com- 3 H. FLASCHKA, Scientia pharmac. 21, 126 [1953]. pounds", Vol. Ill, 547, Eyre Spottiswoode, London 1946.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 2. Experimental of buffer of pn 6. It was interesting to note that murexide exhibits an absorption maximum in the 2.1. Materials used visible region at A = 530 mfi. Further, this wave- Murexide of B.D.H. quality was employed; this was length of maximum absorption was unaffected by purified by the method outlined by DAVIDSON 8, which was found to give a sample of 99.9% purity9. One gram of the commercial sample was dissolved in 900 cm3 of pure distilled water. The filtrate was salted out with 60 gm. of ammonium chloride. The precipitated substance was washed with absolute methyl alcohol, to be free from chloride and later dried at 170° C, when fine reddish brown crystals possessing a green reflex were obtained. The materials employed for the buffers which were due to C 1 a r k s and L u b b s and Sörensen, were Wave length of analytical grade and were specially meant for the purpose. Fig. 1. Absorption spectra of murexide. (Curve 1 refers to The hydrochloric acid used was of B.D.H. Analar 0.125 and curve 2, to 0.0625 mM of murexide; ph 6.0; quality. The hydrobromic acid employed was Merck temp. 25° C.) sample and was free from bromine; the absence of this last was confirmed by testing with potassium iodide the concentration of the substance (cf. curves 1 and starch. The nitric acid used was C.P. analysed and 2, Fig. 1), and by pn of the solution. Detailed product of Basic Synthetic Chemicals Ltd. and was of investigations showed that at a fixed pn, the absorp- 99.9% purity. The neutral salts used were KCl, KBr or, KI and tion at / = 530 m/< varied sensibly linearly with KN03 , all these were Merck samples. the concentration of murexide especially in the range 10~3 to 10~6 M (see Fig. 2). This obser- 2.2. Experimental Procedure vation indicated that the absorption of murexide at I = 530 mfi obeyed roughly the Beer's law; the The decomposition of murexide in the acid solutions corresponding constant had a value of 1.04 • 104 or in buffer mixtures of low pu was studied spectro- photometrically using Unicam S.P. 350 D.G. Spectro- moles-1 litre cm-1. photometer; this had an effective light path of 1 cm. At the first instance, the absorption spectra, in the visible region, of murexide solution of a known concentration was studied; from these results, the characteristic wavelength where the absorption was proportional to the concentration of the substance, was selected. The kinetics of the decomposition of murexide were followed by studying the variation with time of the absorption of murexide, at the above characteristic wavelength.

3. Results

3.1. Absorption Spectra of Murexide

Fig. 1 gives a typical series of results on the ab- Fig. 2. Applicability of B e e r ' s Law to the absorption of sorption of murexide in the visible region; the ab- murexide. scissae refer to the wavelength in millimicrons and 3.2. Time-variation of the Absorption at / = 530 mu the ordinates, to the optical density of the system. In Fig. 1, curve 1 refers to a concentration of at pn < 5 0.125 mM; and curve 2 to 0.0625 mM of murexide; The above data given in Figs. 1 and 2 were these data were obtained with a system containing recorded at pu 6, at which and at higher pn values one volume of murexide solution and one volume the colour and the absorption characteristic of the

8 D.DAVIDSON, J. Amer. diem. Soc. 58. 1821 [1936]. 9 J.H.MOSER U. M.B.WILLIAMS. Analvtic. Chem. 26. 1167 [1954]. system appeared permanent in the sense that they es did not alter appreciably even after 20 — 24 hrs. 0,8 At PH < 6, however, the optical density of the system decreased continuously and progressively with time. t 0,7 \ The results given in Fig. 3 illustrate the variation with time of the optical density at pn = 2.2; in Fig. 3 A curves 1 and 2 refer respectively to the initial con- - centrations of 0.125 and 0.0625 mM of murexide.

0.8 o.t

0.3 - | 0.7 \ fi 0.2 ^0.6

0,1

i i 1 ! ° 0 2 V 6 8 10 12 11 16 Time t in minutes 0.3 Fig. 4. Effect of addition of alkali to the acid reacted murexide solution. (Initial PH of the solution 2.2 (curve B) ; PH 0.2 after addition (at the instant indicated by the arrow) of alkali 8.0 (Curve A) ; A = 530mu.) 0.1 pletely irreversible nature of the reaction but also 0 the dependence of the reaction on the pu or the 0 10 20 30 hydrogen ion concentration (vide infra, 3.5). Time t in minutes

Fig. 3. Time variation of optical density at 1 = 530 mu 3.3. Kinetics of the Reaction: First order nature (PH 2.2; Curve 1 refers to 0.125 and curve 2, to 0.0625 mM of murexide.) Table 1 gives results on the time necessary for half decomposition of the initial concentration of The decrease in the optical density with time was murexide recorded at different pn values. These are accompanied by the disappearance of the characteristic pink-red colour of murexide; at the end of C: c2 H ll order of [M x 103 J [M x 103] [sees.] [sees.] reaction 30 min., the solution of which the data were given by curve 1 (Fig. 3) was absolutely colourless; 2.2 0.125 0.063 4.25 3.825 0.96 neither did it acquire colour when the solution was 2.4 0.125 0.83 6.625 5.125 0.93 2.6 0.10 0.05 4.30 3.20 0.93 made alkaline, indicating that the change respon- 2.6 0.075 0.038 3.60 3.05 0.96 sible for the above variation of the optical density, 2.6 0.05 0.025 3.20 3.00 0.98 was irreversible in nature. This was substantiated, 2.6 0.125 0.063 8.25 7.00 0.96 2.8 0.125 0.063 17.0 12.0 0.91 rather demonstrated in a remarkably clear way, by Mean = 0.95 the data in Fig. 4; these refer to the experiments in which during the progress of the reaction, the pn Table 1. Determination of the order of reaction of the de- of the solution was increased to 8 by addition of composition of murexide in acid solutions. 0.2 cm3 of 4 N sodium hydroxide solution which caused negligible dilution of the solution. It was inter- employed for the determination of the order (n) of esting to note that as soon as the pn was raised, the the reaction by the familiar half-time decomposition reaction leading to the decrease in the optical den- method. The figures in the last column gave an sity of the system was quenched as evidenced by the average value of 0.93 for n, indicating that the observation that the optical density of the solution reaction is roughly first order in nature. It is how- recorded just before the addition of the alkali re- ever to be pointed out that this method is only an mained unaltered with further progress of time. approximate one since the calculation of n involves This observation not only demonstrated, the com- the use of the extrapolated value of the optical density at zero interval of time. That the reaction cording to this equation, the plot log(r'— r) against is first order in nature is, however, substantiated t should be a straight line; then, the slope of the by what follows. line gives the value of the velocity constant. It is There are several methods for examining whether instructive to note from Fig. 5 the sensibly linear or not the reaction is of first order, and to calculate plots of log(r' — r) vs. t obtained from the data the corresponding rate constant, kx, from the read- given in Fig. 3. The value of the velocity constant ings r at time t of some physical property charac- computed from the slope of the line corresponded teristic of the substance undergoing change and to 7.98 • 10~6 min-1 (25° C) for an initial concen- proportional to the concentration thereof. Since the tration of 0.125 mM of murexide at pu = 2.2. optical density of murexide at A = 530 m// obeyed roughly the Beer's law, it appeared justifiable that 3.4. Dependence of the velocity constant on Con- the optical density readings recorded at different centration of Murexide time intervals were proportional to the concentration The data given in Fig. 6 show the time variation of murexide existing in solution; and that the same of optical density of murexide solution of different could be satisfactorily employed for calculating the concentrations; these were obtained by systems con- velocity constant kx . The following is the familiar taining equal volumes of 3.75 mM of HCl acid so- equation for obtaining the first rate constant: lution and of murexide solution of varied concentra- r — a exp (k • t) (1) tions. The concentrations of the solutions with re- spect of murexide have been indicated in Fig. 6. where a is the initial concentration of the substance or the optical density at £ = 0 and r, the same at time t. Since the value of a, as mentioned above, is susceptible for error by extrapolation method, the following equation which is deducible from eq. (1) and does not involve the determination of a, is employed10: r — r = constant • exp (k • t) ; (2) here r is the reading at time t + r where r is a suitably choosen constant interval of time10. Ac-

3,0 I / / Fig. 6. Effect of concentration of murexide on the time varia- 1J tion of optical density at A = 530 mfx. (Curve 1 refers to 0.10; curve 2 to 0.075; curve 3 to 0.05; and curve 4 to h 0.0375 mM of murexide; concentration of HCl: 3.75 mM.) IB

It was interesting -to note that the velocity constant

k1 computed from the slope of the lines log {r — r) 12 1 vs. t given in Fig. 7 varied appreciably with a change in the concentration of murexide. Thus 6 1 0.6 e.g. kx was 9.23 and 10.38 • 10" min" at 0.025 // and 0.075 mM of murexide respectively. kx increased with increase in concentration up to 0.08 mM;

0 1 a further increase in concentration of murexide de- 0 70 20 30 <40 Time in minutes *- creased the constant kt (see Fig. 8). Fig. 5. Determination of the velocity constant of the decomposition of murexide. (Curve 1 refers to 0.125; curve 2 to 3.5. Effect of Strong Acids on the Velocity Constant 0.0625 mM of murexide; pu 2.2.) The influence on the velocity constant kx, of the

]0 E.A.GUGGENHEIM, Philos. Mag. 2. 538 [1926]. decomposition of murexide, of the following acids Vy/ 3/1 y

y / / -

*// / A /

y / /

/ // 12 11 16 18 i • Time t in minutes 10 12 11 16 Time t in minutes Fig. 9. Effect of concentration of HCl on the variation of log(r' — r) with time t. (Curve 1 refers to 10 mM; curve 2 to Fig. 7. Variation of log (r' — r) with time at different concen- 7.5 mM; curve 3 to 6.25 mM and curve 4 to 2.5 mM of HCl; trations of murexide. (Curve 1 refers to 0.125; 2 to 0.10; initial concentration of murexide: 0.25 mM.) 3 to 0.075; 4 to 0.05 and 5 to 0.0375 mM of murexide; concentration of HCl: 3.75 mM.)

| 1.6- /i M

l.o

0ß - f

5 7 9 11 13 to' 0.6, i i Concentration in Motes 1 6 8 10 12 11 t Fig. 8. Variation of velocity constant with concentration of Time in minutes - murexide. Fig. 10. Effect of concentration of HBr on the variation of log(r' — r) with time t. (Curve 1 refers to 10 mM; curve 2 to 7 mM; curve 3 to 5 mM and curve 4 to 3 mM of HBr; initial were examined: hydrochloric, nitric and hydro- concentration of murexide: 0.25 mM.) bromic acid. For this purpose, the procedure adopt- ed was to take a constant volume containing fixed 19 amount of murexide and mix with a known volume / / * '// A of the acid solution such that the final solution I 1'7 /i corresponding to different experiments were of the <5 same volume and contained a fixed amount of murexide but different quantities of HCl, HBr or 1.3 if/}.

HN03. The data in Figs. 9 — 11 give the linear plots 1.1 1 / log (r — r) vs. t referring to HCl, HBr and HN03 respectively. It was interesting to note that with the 0.9 change in the concentration (C) of an acid or the hydrogen ion concentration, the plots varied re- 0.7 gularly, their slope being increased with increase i i i i in C; in other words, the velocity constant in- 8 10 12 11 Time t in minutes creased with C. Thus e.g. kx was 6.25, 16.1 and 6 -1 24.5 • 10~ min at 2, 6 and 10 mM of HNOa . Fig. 11. Effect of concentration of HN03 on the variation of Similar results were obtained with other acids. It log(r' — r) with time t. (Curve 1 refers to 10 mM; curve 2 to 8mM; curve 3 to 6mM; curve 4 to 4 mM and curve 5 to was instructive to note from Fig. 12 that the varia- 2 mM HN03; concentration of murexide: 0.25 mM.) tion of kx with C was linear; kx followed a relation- studied in the presence of only one acid viz. HCl. ship: The reaction mixtures were so prepared that they Äj/min-1 = 2.56 • 10~3 C. (3) had the same quantities murexide and hydrochloric acid, but varying amounts of the salts. A typical Furthermore, the data in Fig. 12 show that the group of results are returned in Figs. 13 and 14. points referring to all the three acids fall roughly, within experimental errors, on the same line indicat- 2.2 ing that eq. (3) governs the effect on the velocity 2j0 constant, of all the three acids; this suggests that -

the anions of the acids have no significant effect on ^r 1,8 the velocity constant. /

1.6 - 2t /

„ I • oßo 2 H 6 8 10 Time t in minutes —

Fig. 14. Influence of different neutral salts on the velocity >r1 i 1 i constant. Concentration of neutral salt: 200 mM; initial con- V 5 6 7 8 centration of murexide: 0.25 mM; concentration of HCl: Concentration in millimoles 20 mM. KCl ... X, KI . .. O, KBr ... •. KNOs ... A-

Fig. 12. Non-variant nature of the velocity constant with different strong acids. HCl ... X, HBr . . . O, HNO:,... A- In these, log (r — r) referring to different experiments were plotted against time t. In Fig. 13, data referring to different concentrations of KCl were 3.6. Effect of Neutral Salts on the Velocity Constant returned while in Fig. 14, the results on the effect The influence of neutral salts like KCl, KBr, KI of all neutral salts of a known concentration were

and KN03 on the decomposition of murexide was given. It was interesting to note that all the points referring to different experiments fell roughly on 22 r the same straight line log (/— r) vs. t; these results support the above deduction (3.5.) that in the process responsible for the decomposition of murexide, the anions have no significant role.

4. Discussion

The colour of murexide (I) appears to be due to the presence of chromophoric groups (two = C = 0 groups in para position, C —N = ), etc. in its mole- cule:

O • NH4 O 2 V 6 8 10 \ Time t in minutes —•> NH—C C—NH

Fig. 13. Influence of the concentration of KCl on the velocity- 0 = C C—N=C c=o constant for the decomposition of murexide. (Concentration of murexide: 0.25 mM; concentration of HCl: 20 mM; open NH—C C—NH circles refer to 400 mM of KCl and closed circles to 200 mM of KCl.) The decolourisation of murexide can be due to the of the inversion of sucrose13; in this case also, k formation of a leuco compound which is known to varies with the concentration (C) of the acids: the get converted back into murexide by K3Fe(CN)6 change in the value of k with C is represented by or by shaking with air11. In the present investiga- the following relationship: tion, however, the colourless solution obtained by Ar/min-1 = 6.95 • 10-3 C • 10fijc (4) reaction of murexide with acid solutions, did not produce, on treatment with K3Fe(CN)6 or/and on where Bj is a constant determined by the anions shaking with air, any pink-red colour characteristic and is 0.28 for CF, 0.35 for Br- and 0.30 for N03~. of murexide and the observed decrease in the optical This observation when analysed on the principle density at 2 = 530m,w, were irreversible in nature of specific interaction of ions, due to Brönsted, (3.2.). It is known 12 that murexide undergoes de- indicated the significant role of the anions of the composition in acid solution to give uramil (II) acids and of neutral salts on the reacting complex and alloxan (III). involved in the inversion of sucrose, which has a single positive charge 13. The finding that eq. (3) NH—CO NH—CO is applicable to the data for the decomposition of

OC CHNH2 CO CO murexide (Fig. 12) and also the data on the effect of neutral salts on the velocity constant (3.6.), HN—CO NH—CO

(II) (III) suggest that the value of B) for CF, Br~, N03~ etc. is zero and that the reacting complex involved in This reaction appears primarily to be a hydrolysis, the decomposition of murexide, as envisaged in the facilitated by acid solutions, similar to the inversion above mechanism, is electrically uncharged in cha- 13 of sucrose . The hydrolysis of murexide appears racter. to proceed as follows: A variation of the first order rate constant with Purpurate ion (anion) + FT-> Purpuric acid, (A) concentration of the substance undergoing change Purpuric acid + H20 —> complex, (B) is not unfamiliar in reactions of the type of acid Complex —> alloxan and uramil. (C) hydrolysis. In the case of inversion of sucrose again 13, a linear increase of the velocity constant k The observation that the reaction follows first order with concentration of sucrose has been observed. law (3.3.) indicates that in the above processes, In the present investigation however, kt increased the reactions leading to the formation of the com- up to 0.08 mM of murexide; a further increase plex are rapid and do not contribute directly to the caused a decrease in the value of k1 ; this observa- observed kinetics. The decomposition of the complex tion is rather interesting and merits further con- (cf. process C) unimolecularly appears chiefly re- sideration. sponsible for the applicability of eq. (2) to the observed data on the time variation of the optical Author's thanks are due to Professor K. S. G. Doss, density of murexide in acid solutions (Fig. 3). D. Sc., F. R. I. C., F. Inst. P., F. A. Sc., Director, Indian It was instructive to refer to the results on the Institute of Sugar Technology, Kanpur, for his interest effect of strong acid on the velocity constant (k) in the work.

11 R.KUHN u. J.C.LYMAN, Ber. dtsch. ehem. Ges. 69 B, 1547 13 E. GUGGENHEIM u. L. A. WISEMAN, Proc. Roy. Soc. [London] [1936]. 203, 17 [1950]. 12 Cf. ref. 6.