Polarographic Studies on Reductones and α-Keto Acids

I. L-Ascorbic acid (AA) showsan oxidation wave at pH less than 8 and two in a more alkaline range. After standing of AA in alkaline solution, the third oxidation wave has been observed. Dehydro-L-ascorbic acid (DAA) has been also tested in alkaline solution. The mechanism of the electrode oxidation of AA and its decomposition in alkaline solution are discussed. The reduction wave of DAA has been obtained in pH 2.2 to 5.0, in which the half-wave potential (π_<1/2>) varies from -0.35 to -0.48V. (vs N.C.E.) The wave height is about 1/1000 of its expected diffusion current at room temperature. The wave has all the typical characteristics of the kinetic current. The shape of the wave is strongly deformed by adding small amounts of surfaceactive substance such as gelatin. The reduction product of DAA at the dropping mercury electrode has been polarographically proved to be AA. Discussions are made on the configuration of the inactive form of DAA and the reversibility of AA and DAA system. II. Alloxan, dehydro-reductic acid (DRA), and mesoxalaldehyde (MOA) show reduction waves which are similar to that of DAA. π_<1/2>s of alloxan, DRA, DAA and MOA in pH 3.5 are -0.05, -0.27, -0.42 and -0.35V, respectively. Of these four substances, the lower the height of the reduction wave, the greater both the shift of the π_<1/2> of the anodic wave of the corresponding reduced form from the standard oxidation-reduction potential (π_0') of the system, and the temperature coefficient of the height of the reduction wave. Alloxan has been proved to be polarographically reversible by the controlled potential electrolysis, and the π_<1/2> of the reduction wave coincides with that of the oxidation wave of dialuric acid, while no such observations have been made with other three. III. The polarographic behavior of DAA, DRA, alloxan, MOA and 2, 3-diketogulonic acid (DGA) in acid buffer solutions containing o-phenylenediamine (OPD), has been investigated. DAA shows three diffusion controlled reduction waves, whose π_<1/2>s at pH 3.60 are -0.26, -0.44 and -0.67V, respectively. The limiting currents of the three waves depend on the time after the addition of OPD, the concentration of OPD, pH and the temperature. When the electrolytic solution contains both AA and DAA, the oxidation wave of AA can be obtained independently of the three waves for DAA in one polarogram. DGA shows two reduction waves whose π_<1/2>s at pH 3.44 are -0.27 and -0.40V., respectively. Alloxan and DRA show single well denned reduction waves, whose π_<1/2>s at pH 3.60 are -0.32 and -0.19V, respectively. MOA shows no well denned reduction wave in acid media. Since the linear relationship between the limiting current and concentration has been obtained for DAA (the third wave), DRA and alloxan, these polarographic phenomena have been applied to the simultaneous determination of AA and DAA, and to that of RA and DRA and further to that of AA and RA. The method is also applicable to the determination of alloxan, but not to those of MOA (or triose-reductone) and DGA, none of which, however, seriously interfere with the determination of DAA (or AA) by this polarographic OPD method. IV. The pH-dependence of the limiting currents of mesoxalic acid, dihydroxytartaric acid, phenylglyoxylic acid, trimethylpyruvic acid, phenylpyruvic acid, oxalacetic acid, pyruvic acid and α-ketoglutaric acid has been investigated. The limiting currents of the first reduction waves of these acids have their maximum values in pH near 4, and in pH below 4 they show appreciable decreases with decreasing pH. Some of the acids show a reincrease of the limiting current in pH below 1. This characteristic behavior has been accounted for by hydration of carbonyl group and in some cases by enolization besides hydration, comparing polarographic results with spectrophotometric measurements. A solw establishment of the keto-enol tautomeric equilibria can be followed in the case of phenylpyruvic acid either polarographically or spectrophotometrically. The reaction rate constants of ketonization and enolization of the acid have been obtained to be k_<keto>=6.52×10^<-4>sec.^<-1> and k_<enol>=7.4×10^<-5>sec.^<-1> in 0.1N hydrochloric acid aqueous solution containing 196 alcohol at 25℃. It is pointed out that the rate constants of dissociation and recombination of the above mentioned acids previously reported by other authors, should be reinvestigated. The question whether the enol-form participates in the polarographic behavior of pyruvic acid is still open. V. The limiting current of trimethylpyruvic acid and its UV-absorption spectra have been studied in relation with hydration of carbonyl group of undissociated acid. pK_a Value for the apparent dissociation constant of trimethylpyruvic acid has been spectrophotometrically obtained to be 1.52 at 25℃. Hydration constant of the undissociated acid (K^η_h=[AH_<aq>]/[AH]) has been kinetically obtained to be 0.46 at 25℃. The rate constant of dehydration of hydrated trimethylpyruvic acid in buffer solution of pH about 5 was 2.8×10^<-2>sec.^<-1> at 25℃. From the temperature dependence of the hydration constant, the heat of hydration of the acid has been found to be -8.2 kcal. A quantitative interpretation of the pH-dependence of the limiting current has been attempted by deriving the equation involving the hydration and dissociation factors. The agreement of the theoretical curve with the experimental is good, except for the pH range less than 0.5, where acid catalyzed dehydration should be taken into consideration. The rate constants of the dissociation and recombination of trimethylpyruvic acid have been found to be k_d=6.0×10^5sec.^<-1> and k_r=1.4×10^7 l.mol.^<-1>sec.^<-1