Tab!e V. Results of Interaction between Representative Indole Derivatives and Ethchlorvynol Name LSD maleate Ergotrate maleate Reserpine Methysergide Rescinnamine Yohimbine Meprobamate : Reference
Color in chloroform Initially Later Violet Light blue Pink Violet Violet Light brown Green
Green-blue Green-blue Violet Green-blue Blue Violet Dark green
Indole Derivatives. Table V shows the colors obtained for a number of indole-containing drugs. LSD, as well as other n-substituted lysergic acid amides produced a violet color initially during the reaction but changed to a more stable bluish green. Once a color was established on addition of 95 ethanol, it remained stable during the 5-minute experimental period. ACKNOWLEDGMENT
We are grateful for the excellent technical assistance of Gale Rosenquist and for helpful discussions with Edet E. Inwang. RECEIVED for review March 30, 1972. Accepted August 21, 1972.
New Kinetic Method for Determination of Ultramicro Quantities of Organic Substances Determination of Amino Acids (Glycine, DL-Serine, DL-Phenylalanine, DL-Glutamic Acid, and L-Arginine) T. J. JanjiC and G . A. MilovanoviC Chemical Institute, Faculty of Sciences, University of Belgrade, Belgrade, Yugoslavia A NEW KINETIC method for the determination of ultramicro quantities of amino acids is described in this paper. The catalytic activity of copper in the reaction of oxidation of pyrocatechol violet by hydrogen peroxide has been found to decrease in the presence of ultramicro quantities of amino acids, because of the formation of 1:1 complexes. Since the decrease in catalytic activity turned out to be proportional to the quantity of amino acid present, a method whereby amino to acids can be determined in concentrations from 2.0 x 8.0 X 10-6Mhas been developed. There are many detailed kinetic studies concerning the determination of ultramicro quantities of inorganic ions ( I ) . In contrast, there is a considerably smaller number of reports dealing with kinetic methods for the determination of ultramicro quantities of organic compounds. The best investigated are the enzyme-catalyzed reactions that have been used for the determination of enzymes themselves (2-6), substrates (7-10), activators ( I I ) , and inhibitors (12, 13).
In order to extend the kinetic methods of analysis to the field of organic compounds, we have decided to investigate changes (decrease or increase) in the rate of some simple metal-catalyzed reactions caused by changes in the catalytic activity of metal ion, due to the formation of metal complexes with organic substance added. We have anticipated that the change in catalytic activity would be dependent on the quantity of the complex formed, which could provide a basis for evaluating the quantity of the organic substance present. It should be mentioned that such an idea was first put forward by Yatsimirskii (14). However, this author merely pointed out that the complex should be stable and catalytically inactive, but did not cite any experimental data in support of it. Generally speaking the reversible reaction of the formation of 1:l complex between a metal catalyst and a compound capable of coordinating it can be presented by the following general reaction scheme:
(1) K. B. Yatsimirskii, “Kineticheskie Metodi Analiza,” I1 Izd.,
M + L ~ M L
Izdatelstvo “Khimia,” Moscow, 1967. (2) W. J. Blaedel and G. P. Hicks, Anal. Biochem., 4,476 (1962). (3) W. H. Marsh, B. Fingerhut, and E. Kirsh, Clin. Chem., 5, 119 (1959). (4) M. K. Schwartz, G. Kessler, and 0. Bodansky, Ann. N . Y . Acad. Sci., 87,616(1960). ( 5 ) G. D. Winter, ibid., p 875. (6) B. Fingerhut, R. Feryola, W. H. Marsh, and J. B. Levine. ibid., 102,137(1962). (7) . , W. J. Blaedel and G. P. Hicks. in “Advances in Analytical Chemistry and Instrumentation,” Vol. 3, C. N. Reilley, Ed., Interscience, New York, N.Y., 1964, p 105. (8) H. V. Malmstadt and T. P. Hadjiioannou, ANAL.CHEM., 34, 455 (1962). (9) Zbid.,35: 14(1963). (10) 0. N. Kramer, P. L. Cannon, and G. G. Guilbault, ibid., 34, 842 (1962). (11) G. P. Hicks, Ph.D. thesis, University of Wisconsin, Madison,
where M is the catalyst, L is the ligand added in a subequivalent quantity in relation to catalyst M to prevent the formation of complexes with higher ligand-metal ratios; ML is the 1:1 complex possessing lower or higher catalytic activity than catalyst M. M indicates the catalyst in its active form, no matter whether it is a hydrated metal ion or any other species that can be formed in solution, or a mixture of them. Therefore, it follows that M L can have a more complex composition, too. L represents a ligand in all its forms present in a n acidbase equilibrium system. Considering the complexity of such systems, charges of ions are omitted. Applying the law of mass action to Reaction 1 and denoting the total concentration of each component as [MI, [ML], and [L] we obtain:
Wis.. 1963. (12) H. W. Linde. ANAL.CHEW.31. 2092 (1959). (13) 0. N. Kramer, P. L. Cannon, and GI G. Guilbault, ibid., 34, 1437 (1962).
390
(14) K. B. Yatsimirskii, “Kineticheskie Metodi Analiza,” I1 Izd., Izdatelstvo “Khimia,” Moscow, 1967, p 92.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
( K is the apparent equilibrium constant at a constant pH value and constant concentrations of all remaining components that could take part in the composition of species M and ML). Since tMlo
=
[Llo
=
+ [MU L I + [MLI
[MI
(3)
(4)
([MI, is the initial concentration of component M, and [L], is the initial concentration of component L), it follows from Equations 2,3, and 4: K[M12
+ [Ml(1 + KLIo - K M o f
- [MI0 = 0
(5)
The limiting values of Equation 5 show: lim[M]
=
[MIo
K+O
-
lim [MI K-.
=
(The second solution lim [MI
[MI0 - [LIo =
K-+-
(7)
0 is meaningless.)
As shown by Equation 6, no complexation occurs, in the first case, while, according to Equation 7, the entire quantity of the added ligand, in subequivalent quantity, is coordinated. In practical work, in the case of sufficiently large K values and within a definite range of total ligand concentration, it is reasonable to expect with a high degree of accuracy that the ligand is completely coordinated (region of analytical application). In such a case it is possible to determine the ligand concentration in solution indirectly applying kinetic methods of analysis. As in this work for that purpose, tangent method ( I ) is applied and as indicator reaction the loss of color of the indicator dye, which was followed spectrophotometrically, is chosen, we shall consider here such a special case, too. There is a following relationship between f g a (slope of the straight line from diagram: logarithm of absorbance of the solution vs. time), [MI and [ML], if both M and ML components are catalytically active: tga = -0.434 ki[M]P,’
- 0.434 k2[ML]Pc”
(8)
(kiand k2 are the apparent rate constants, while P,’ and P,“ denote the products, or even some more complex functions, of the concentrations of remaining components participating in the reaction). Suppose that above mentioned requirements are to a sufficient extent satisfied; it follows from Equations 3,7, and 8 that tga
-0.434 ki[M]oP,’
Figure 1. Dependence of - fgcu on [L], when complex ML is catalytically inactive (a), and when it is catalytically less active than catalyst M (b)
+ 0.434 ki[L]oP,’
-
- 0.434 kz[L]oP,”
(9)
According to Equation 9, one can see that tga will be a linear function of [LI0independently of whether the complex ML is catalytically more active or less active than M, or catalytically inactive. The dependence of - f g a on [LI0 when complex ML is catalytically inactive (a), and when it is catalytically less active than catalyst M (b) is given in Figure 1. To confirm the theoretical considerations, we have studied the catalytic oxidation of pyrocatechol violet by hydrogen peroxide in the presence of copper as a catalyst, and amino acids as ligands. The catalytic reaction with copper as a catalyst was chosen because copper(I1) ion manifests a high catalytic activity and a marked tendency toward coordination as a d9 system. The oxidation reaction of pyrocatechol
35
1
la
.
-t@xlO
0.5 . I
0
02
04
06
08
IO
12
-
AMINO ACID CONCENTRATION [ MxlC’)
Figure 2. Dependence of - f g a on amino acid concentration in oxidation of pyrocatechol violet by hydrogen peroxide in the presence of copper Initial concentrations: pyrocatechol violet, 4.1 X 10-6M; borate buffer, 2.0 X 10-2M; hydrogen peroxide, 17.5 X 10-2M; and copper(I1) perchlorate, 1.0 X 10-6M (1) glycine, (2) DL-glutamic aicd, (3) L-arginine, (4) DL-phenylalanine,(5) DL-serine
violet by hydrogen peroxide in the presence of copper as a catalyst was used first by Birmantas and Jasinskiene (IS) for determining ultramicro quantities of copper in neutral solutions. We carried out this reaction in a borate buffer solution at pH 8.65, since amino acids in neutral solutions do not exhibit any effect on this reaction. (15) J. Birmantas and E. Jasinskiene, “Lietuvos TSR Aukstuju Mokyklu Mokolo Darbai,” Chem. Chem. Technol., 6, 5-11 (1965); Chem. Abstr., 64,1337~(1966).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
391
Table I. Rate Constants for Oxidation of Pyrocatechol Violet by Hydrogen Peroxide in the Presence of Copper as a Catalyst pH - tga
x
102 0 50 1.33 2.27 2.85 0.68 0.78 0.57 0.43 0.27 0.14 0.95 0.57 0.47
=
8.65;
1.1 =
0.1 (NaC104); t = 25 i 0.1 "C
[CUI 105
[Ha02] X 102
molesil.
moles/l.
0.20 0.50 0.80 1 .OO 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
17.5 17.5 17.5 17.5 17.5 21 .o 14.0 10.5 7.0 3.5 17.5 17.5 17.5
x
[Borate] kl X X 102 1. mole-' molesil. min-' 2.00 2.00 2.00 2.00 4.00 4.00 4.00 4.00 4.00 4.00 3.00 5.00 6.00
0.64 0.69 0.73 0.73 0.71 0.69 0.73 0.73 0.69 0.73 0.73 0.73 0.73
Mean value: kl = 0.71 i 0.01 X lo3. Result for kl is given as E , = M -i. F,, where M is the mean value of the rate constant and F, is the standard deviation of the mean. Table 11. Rate Constants for Oxidation of Pyrocatechol Violet by Hydrogen Peroxide in the Presence of Copper-Glycine 1 : 1 Complex as a Catalyst pH = 8.65;
I.1
= 0.1 (NaC104);
[CuGlyc]
x
[H202]
x
101
- tga 102
moles/l.
moles/l.
1.87 1.50 0.93 0.38 0.92 0.32 0.17 0.08 0.25 0.25 0.55 0.40
1 .OO 0.80 0.50 0.20 0.50 0.50 0.50 0.50 0.30 0.50 0.50 0.50
17.5 17.5 17.5 17.5 17.5 14.0 7.0 3.5 17.5 17.5 17.5 17.5
x
105
t = 25 I O . 1 "C [Borate] ka X x 102 1. mole-' molesil. min-1 2.00 0.48 2.00 0.48 2.00 0.48 2.00 0.50 2.00 0.48 4.00 0.41 4.00 0.44 4.00 0.46 4.00 0.44 6.00 0.39 3.00 0.44 4.00 0.41
Mean value: k 2 = 0.45 i 0.01 X lo3. Result for k2is given as E, = M =tF,, where M is the mean value of the rate constant and F, is the standard deviation of the mean.
EXPERIMENTAL
The reaction rate of oxidation of pyrocatechol violet by hydrogen peroxide was followed spectrophotometrically. The absorbance of the solution was measured at the wavelength of 500 nm which corresponds to the absorption maximum of pyrocatechol violet in borate buffer at pH 8.65. The readings were done on a Beckman Model D U quartz spectrophotometer with a device for thermostating in I-cm cells every minute during 10 minutes from the start of the reaction. The solutions were thermostated at 25 i 0.1 "C before the beginning of the reaction. A radiometer 4c pHmeter was used for measuring the pH values of the solutions. Analytical grade chemicals and redistilled water were used for the preparation of all solutions. A quartz glass BiDestillier-Aparat (D.B.P. 1027147), manufactured by QuartzSchmelze GmbH, Wiesbaden-Biebriech, was used for the distillation of water. The standard 0.26M solution of copper(I1) perchlorate was obtained by dissolving basic copper(I1) carbonate (Carlo Erba) in excess of perchloric acid and diluting to vol392
ume with 1 X 10-3N HClOd to prevent adsorption on glass *+ ions. The copper content and protolysis of [CU(HQO)P,] was determined electrogravimetrically. The solution of pyrocatechol violet (Kemika) was 1 x 10-3M, and that of hydrogen peroxide (Merck) 9.8M. The borate buffer H[B(OH)d/[B(OH)4]- (0.1M) was obtained by mixing NasB40i.10HzO (Reanal) and NaOH. All investigations were carried out at a constant ionic strength I.( = 0.1 maintained through the addition of sodium perchlorate solution. The 0.0250M stock solutions of amino acids were prepared by dissolving appropriate amount of glycine (Kemika), DL-serine (BDH), DL-phenylalanine (BDH), DL-glutamic acid (BDH), and L-arginine (Fluka) previously dried at 105 "C. Standard solutions were prepared by the dilution of the aliquots of stock solutions with perchloric acid (1 X 10-3N). The reaction was carried out in the fol!owing way: In a reaction-mixture vessel with three compartments, the solutions of pyrocatechol vlolet, copper(I1) perchlorate, and amino acid were measured into one compartment, buffer into the second, and hydrogen peroxide and water (total volume 25 ml) into the third compartment. The vessel was thermostated and the reaction was started by vigorous mixing. The reaction solution was put into a cell and the absorbance was measured spectrophotometrically each minute. All vessels before use were washed with 6N HC1 and then rinsed several times with running, distilled, and, finally, with redistilled water. RESULTS AND DISCUSSION
Following the reaction rate of the oxidation of pyrocatechol violet by hydrogen peroxide in the presence of copper as a catalyst and amino acids (glycine, DL-serine,DL-phenylalanine, DL-glutamic acid, and L-arginine), it was found that amino acids exhibit an inhibitory effect on this reaction due to the formation of 1: 1 copper-amino acid complexes possessing lower catalytic activity than copper. The experimentally determined functional dependence of -rga = ,f([amino acidlo) is presented in Figure 2. It shows that in all the amino acids examined, this dependence is linear for [amino acidlo 50.8 x loF5M , which can be applied to the determination of glycine, DL-Serine, DL-phenylalanine, DLglytamic acid, and L-arginine. To find the optimal conditions for the determination of amino acids, we have examined the kinetics of oxidation of pyrocatechol violet by hydrogen peroxide in the presence of copper, as well as in the presence of copper-glycine 1 : 1 complex. The kinetics of oxidation of pyrocatechol violet by hydrogen peroxide in the presence of copper was determined in the usual way-i.e., by following the changes of tga in dependence of the concentration of the examined component, at a constant concentration of all other components which take part in the reaction. This was found, in the pH range investigated (8.35-9.85), to be consistent with the following equation: dx dt
- - = k,[P~][Cu][H,O,][Borate!-~
(10)
([Pv] = pyrocatechol violet concentration, [Borate] = total buffer concentration; charge of Cu is omitted for the same reason as in the case of M.) The kinetics of oxidation of pyrocatechol violet by hydrogen peroxide in the presence of copper-glycine 1: 1 complex could not be determined directly, because of the possibility of disproportion of this complex (in the case of equivalent concentrations of metal ion and glycine) or the formation of a 1 :2 complex (in the case of glycine in excess).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
Therefore, we carried out analogous investigations as in the previous case, with solutions that contained copper and glycine in a molar ratio 2: 1. We assumed that the concentrations of metal ion and the 1: 1 complex in the solution were equal and that two parallel catalytic reactions were taking place. As tga of the metal catalyzed reaction as well as the total value of tga could be determined experimentally, tga of the complex catalyzed reaction was obtained from their difference. In this way, the following kinetic expression for the oxidation reaction of pyrocatechol violet by hydrogen peroxide in the presence of the copper-glycine 1: 1 complex as a catalyst was found:
Table 111. Determination of Ultramicro Quantities of Amino Acids by Tangent Method Found Taken Re1 rnoles/l. No. of stand moles/l. x 106 x 106 detns dev, Amino acid 2.07 f 0.01 10 4.8 2.00 Glycine
m-Serine
DL-Phenylalanine
dx dt
- - = k~[Pv][CuGlycl[H2021[Boratel-i (charge of CuGlyc is omitted for the same reason as in the case of ML.) According to Equations 10 and 11, the apparent rate constants of these reaction (kl and kz) were calculated and the values obtained are presented in Tables I and 11. The data in the tables show that the mean value for kl is 0.71 i 0.01 X lo3, whereas the mean value for kS is 0.45 i 0.01 X lo3. The values of k? for the other amino acid complexes are: for DL-serine complex, 0.36 i 0.01 X lo3;for DL-phenylalanine complex, 0.38 + 0.02 x l o 3 ; for DL-glutamic acid complex, 0.45 Ilt 0.01 X l o 3 ;and for L-arginine complex, 0.44 i 0.01 x 103. As can be seen kinetic Expressions 10 and 11 differ only in their rate constants, which means that any change in the concentrations of the components participating in the reaction could not increase the sensitivity towards glycine. It can be concluded that the optimal conditions for the determination of glycine are the same as those for the determination of copper. On the basis of all these results, we applied the reaction of oxidation of pyrocatechol violet by hydrogen peroxide in the presence of copper as a catalyst for the determination of ultramicro quantities of amino acids. The initial concentrations of components in the reaction solution were: pyrocatechol violet, 4.1 x 10-6M; borate buffer, 2.0 x 10-*M; hydrogen peroxide, 1.75 x 10-lM; copper(I1) perchlorate, to 10 X 1.0 x 10-6M; and amino acids, from 1.0 x 10-fiM. The results obtained are presented in Table 111. The table shows that, by using the described method, we have determined the amino acids in concentrations ranging from
DL-Glutamic acid
L- Arginine
4.00 6.00 8 .OO 2.00 4.00 6.00 8 .OO 2.00 4.00 6.00 8.00 2.00 4.00 6.00 8.00 2.00 4.00 6.00 8 .OO
4.10 i 0.14 5.90 Ilt 0.08 7.82 i 0.22 1 .97 f 0.16 3.83 Ilt 0.15 5.96 Ilt 0.35 7.78 i 0.15 1.99Ilt0.18 4.27 i 0.27 5.93 Ilt 0.23 8.13 Ilt 0.25 1.82i0.16 3.91 f 0.04 5.76 i 0.49 7.50 i 0.15 2.08 Ilt 0.19 3.75 i 0.22 5.96 i 0.28 7.99 + 0.53
3 3 10 10 3 3 10 10 3 3 10 10 3 3 10 10 3 3 10
3.4 1.3 2.8 8.1 3.9 5.8 1.9 9.0 6.3 3.8 3.1 9.1 10.2 8.6 2.0 9.1 5.8 4.7 6.6
2.0 x 10-6M (with standard deviation of 4.8% for glycine, 8.1 % for Dpserine, 9.0% for DL-phenylalanine, 8.7% for
DL-glutamic acid, and 9.1 % for L-arginine) to 8.0 x 1OPfiM (with standard deviation of 2.8% for glycine, 1.9% for DLserine, 3.1 % for DL-phenylalanine, 2.0% for DL-glutamic acid, and 6.6% for L-arginine). Taking into consideration the small difference between k , and ks,it is understandable that all measurements had to be performed under very constant conditions. The application of the proposed method for determining some other organic substances important in pharmacology (histamine and antihistaminic agents, vitamins, antibiotics, bacteriostatics, fungicides, and others) is in progress. These determinations are based on decrease or increase in the catalytic activity of cobalt and manganese in oxidation reaction of alizarin S and pyrocatechol violet by hydrogen peroxide. RECEIVED fur review April 11, 1972. Accepted August 30, 1972.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
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393
