Easy Experiments for Detection of Chromium Intermediates S. Signorella, M. Rizzotto, M. Mulero, S. Garcia, M. Frascaroli, and L. F. ~ a l a ' Area Inorganica, Facultad de Ciencias Bioquimicas (UNR)Suipacha 531 (2000) Rosario, Argentina Detection of reaction intermediates is an important matter when a kinetic experiment is carried out, to reinforce the mechanism derived from the rate law. However, this task may be difficult when the transient species are very short-lived, and under kinetic conditions they may not be observed. If this is so, conditions under which detection would be feasible will have to be found out, and students should be taught to do it. Herein oxidations of organic substrates by potassium dichromate in aqueous solution have been chosen as model systems, since both Crv and Crvl complexes involved in these reactions ( I ) may be detected easily. When CrV1is mixed with biologically related molecules in aqueous solution, growth and decay of Crm and CrVintermediates are observed, with "stability" ascribed to complex formation with substrate (2).Besides, stable CrVcompounds with ahydroxy-acids having a tertiary alwhol group have been isolated and characterized (3).Identification of CrVintermediates has led to the consideration that such species may he involved in the molecular processes leading to the onset of cancer formation, and they are likely candidates for the ultimate carcinogenic forms of carcinogenic chromium compounds (4). Chromium(V) compounds may be detected by EPR or from their characteristic absorption band around 750 nm, where the other chromium species do not interfere; whereas, CrV-esten may be detected spectrophotometrically at 410 nm. 'To whom the correspondence should be addressed.
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Herein is prevented a practical and easy exercise that is helnful to illustrnte an aoolication of EPR and soectmohotoketric techniques to &ect intermediates in'a with probable biological implications. Experimental Oxalic acid (Timper,AR), gluconic acid (Sigma grade), Lrhamnose (Sigma grade), 2-hydroxy-3-methyl-butanoic acid (Aldrich grade) and potassium dichromate (Ciccarelli
Fgure 1. Dtfferental ws~blespectra for tne react on ofcrV' with oxallc acd at several tmes. A A= A,, - A,, where A 1s the absorbance of a salmon of crJ w ~ t hthe same concernranon Jseo in the reanlon mlxture.
Conditions Required for Easy Detection of crV EPR Signals and grade) were used without further purification. Perthe Relative First-Order Maxima Uh = chloric acid solutions were ~ r e ~ a r from e d 70% ~ e r hma~(substrate)lh~~,(rhamnose)) Observed in the Reaction of chloric acid (Merck, ARI. crV'with Various Oraanic Substrates at 25 'Ca Aaueous solutions containine various Crw-substrate mole ratios were preparA by addition of po[HC104] AcH hr g tassium dichromate solutions to water containing Substrate [S]x lo2 [c?] x the required amount of substrate and adjusting the (M) 10(M) (M) (%) (cmlcm) pH by addition of perchloric acid to the final values. gluconic acid 2.5 10.0 0.125 25 1.8 1.977
. .
W a r n i n g : AU Crw compounds are toxic and poteutial carcinogens. Chromic acids are extremely corrosive and should be handled only by individuals wearing appropriate protective eyewear and protective clothing.
0xalic acid
5.0
2-hydr0xy-3-methyl-2.3 bulyric acid L-rhamnose 77.5
20.0 7.75
43
1.0
11.9 1.969 6.5 1.977 2.6 1.977
3400.0 0.25 1.0 1.977 The EPR signal for Crv was studied with a Bruker 41at -11s should be employed for aqueous solutions to prevent losing the microwave SDectroDhotometermeasureER-200 soectrometer, S'gnal' ments were made with ~ i l f k Response d UV-VIS spectrometer. An excess of organic substrate was used for all EPR and spectrophotometric measurements. Under these and hydroxy-carboxylicacids (5), according to the following conditions the four reactions stoichiometries are represequence: sented by eqs 14: 2crw + 2crN + ~ r "+ C? + crm (5) 8 F + 3C6Hlz07,+ WCr04Since reactive intermediates cannot be detected "easily", = 3C5HlOO5 + 3C02+ 2cr1" + 8Hz0 (1) the present experiments focus on detecting Cr"' and CrV 8W + 3Cz04Hz+ 2HCr04intermediates. = 6C02+ 2cr1" + 8H20 (2) Detection of Chromic Ester Intermediate 8H' + 3C5H1003+ 2HCrOp I t is well documented that Cr"' has a large tendency to = 3COz + 3C4H80+~ c+ P 8Hz0 (3) form chromic esters with substrates containing oxygen 8Ht + C6H1205+ 2HCrOp atoms (8).This Crw-substrate complex formation always = C5H1005 'Oz+ 2crm+ 6Hz0 (4) precedes Cr"' decay according to eqs 6,7: Initial one- (5).two- (6).or three- (7)electron reductions of Cr"' have b&n for chr&c oxidations of organic substrates. However, it is generally acknowledged that a n initial two-electron reduction occurs for carboxylic
a
+
Differential wavelength scanning of the reaction between 350 and 500 n m a t several times, shows a maximum (AA,,) a t h = 410 nm. For carboxylic acids this maximum increases and decreases with time (Fig. 1 shows typical curves for oxalic acid), while for L-rhamnose (and for other aldoses) the maximum always decreases. If the CrW-substrate complexes obey Beer's Law, then the relative esterstabilities indicated by Figure 1should be:
Besides, positive values in abs are reached when the ligand is an a-hydroxy acid, due to the presence of the carboxylate group, a hard base t h a t is expected to bind strongly with a hard acid a s Cr"'. When the carboxylic group is replaced by a n hemiacetal, the "stability" of the ester complex decreases (kl> K ) . Several works support these ideas (9).
Figure 2. EPR spectra for complexes of c r Vwjlh (a)gluconic acid, (b) oxalic acio, (c) 2-hydroxy-3-metnyl-butanolc acla, an0 (dl .-rharn. nose.
Detection of crV by EPR Measurements The table shows the concentrations required for easy detection of a CrVEPR signal in each reaction. Diluted carboxylic acid solutions when treated with Cr"' yielded CrV in large enough quantity to be detected, but brhamnose and Cr"' gave a n EPR signal only when saturated solutions were used (Fig. 2). Gluconic acid-Crv and 2-hydroxy-3-methyl-butanoic acid-Crv spectra are dominated by a sharp band a t g = 1.977 due to CrV with its four peaks (9.55% abundance, I = 3/21hyperfine bands a t 18.3 G spacing. Under all Volume 69 Number 7 July 1992
579
0.06
n (cml
*be
20
responding EPR si al can be observed. In all other cases, when the rate of CrTdecay is higher than that of its formation (k3> kz),no EPR signal could be observed. When two peaks are observed, eqs 14 and 15 should be added, and the former suppositions may be applied to either of these cases. K" C 9 + xs 2crvsx (Cz) (14)
Figure 3. (a)Ftrsl derivative he~ghtof the EPR signal of 2-hydroxy-3methyl-butanoic a c ~ b ~comp r " ex at differenttimes. Spectrophotometr clime scanning forthe reanion of CI" wlth ( 0 )gluconlc acld, (c) oxal~caco, and (d) 2-hydroxy-3-methyl-butanoicac d, at 750 nm. experimental conditions no additional bands were observed. This band decays with time, but it is still observable aRer a t least 20 min. Atypical curve of the first derivative height versus time is sh& in Figure 3. The oxalic acid-Crv spectrum exhibits two sharp bands at g = 1.977 and 1.970, respectively, due to CrV(Fig. 2). Rates of decay of the two bands are comparable, and they still are observable after at least 16 min. The brhamnoseCrv spectrum exhibits a shar band at . 2). e = 1.977and a broad band a t p = 1.969. due to Crg (Fie. The sharp band decays with tike, and is observable && 15 min. However, the smaller CrVband is distorted by the Crnl band, preventing the independent monitoring of its behavior. The presence oftwo EPR peaks for the last two reactions (eos 3 and 4 ) means that two different intermediate CrV c 0 ~ ~ 1 e xare e s formed; while in the former cases (eqs 1and 2) only one CrVspecies is mainly formed. Under the same detection conditions it is possible to compare the concentrations of the main Crv species formed in the reactions studied. If CrVreacts as it is formed, relative formation and decay rates determine its actual concentration. So, in the course of the Crv-ligand interaction, CrVis formed and takes part further both in coordination and redox crW+So c r " ' ~ (8) c~"'s -+ crN + P (9) crN + s + crm + S' (10)
-
Relative first derivative maxima for bands due to the main CrV species are shown in the table. The lowest value is observed for brhamnose-Crv. even when reactants saturated solutions were used. his means that this complex should have a lower effective concentration than those formed by carboxylic acids. These results may be interpreted f?om eas 11-13, when k~ and kr have similar values. In this way, crhamn&e-C!rv complex would represent a limiting case between those in which CrVmight be detected or not. Besides, an additional "stabilization"of CrVcomplexes by carboxylic groups (higher values of If in eq 12) would support the different behavior between aldoses and hydroxycarboxylic acids.
..
Measurements Detection of crVbv S~ectro~hotometric The same concentrations of reactants shown in the table were used for spectrophotometric measurements. Molar absomtivities of Crv comolexes at 750 nm are small (10). requihng a 10-cm cell Time scanning of the reaction mixtures at 750 nm showed tvoical curves for 2-hvdroxv-Smethyl-butanoic acid, oxalii kid, and gluconic~acid-&v intermediates complexes (Fig. 3). Unfortunately, the ~ r h a m n o s e C complex r~ cannot be detected by this technique, even though a persistent dark brown color, characteristic of Crvcomplexes,was observed. A very low value of the wmplex molar absorptivity may explain this result. Conclusion In the well-known oxidative degradations of organic substrates with Cr"', students need to learn an appreciation for the chemistry of the various oxidation statets) [of Crl involved. In these experiments, students may recognize reaction intermediates; and by using EPR and spectrophotometric techniques to complement kinetic measurements, they can possibly modify mechanisms previously proposed. Acknowledgment The authors thank CONICET (National Research Council of Argentina) and the University of Rosario for financial support. Literature .Cited 1. M i t e ~ % M.; Batcheu, P.R C w r C k m Rev. 1R3,61.24-212. 2. Goodgame, D. M.;Heyman,P 8.: Hathway, D . E.Polykdron 1882.5.497-499.
In cases where the rate of process (11)and the equilib-
rium constant IC of process (12) are high enough, the cor-
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Journal of Chemical Education
3. Krurnpole, M.; Roeek, J. Imrg Synth. 1980.20.6365. 4. Wetterhahan, J. K.J Am. C k m Soe. 1982,104,674475. 5. Kwmg, D. W ;. Penninpton,D.E. I n w g C k m . 1984,23,252&2532. J. hog.C h . lW6,%,2T4C6. H~ht,G.P.;Jursich,G.M.;Kelsa,M.T.;Mernll,P 2746. 7. Ramesh. S.; Mahapatm, S. N.; Liu. J. H.; R o d , J. J . A m C k m Soe 1881, la?. 51724175. 6. McCann, J.P.;MeAuley.A J Chem Soc M t o n 1976,785-TOL. 9. Colton, R. Cmr. C k m . Re". 1988.90.1-27. 10. Ip,D.;Roak, J. J . A m C h . Soe. lW9,101.63114319.
