Abstract
It was found in the iodometric titration of pyruvate that a higher reaction temperature decreased the iodine consumption, and the maximum consumption was observed at 0°C under the similar conditions of the Wieland method (Fig. 1). The determined values of pyruvate obtained by the method were compared with those obtained by a modified 2, 4 dinitrophenylhydrazine (2, 4-D) gravimetric procedure of the Simon method. The amount of 2, 4-dinitrophenylhydrazone (2, 4-DZ) dissolved in the reaction mixture was investigated. The results indicated that the determined value given by the iodometric titration might be corrected by multiplying the correction factor, 1.011. The outline of the established procedure is as follows.
Weigh precisely about 250 mg of sample pyruvate (W mg), dissolve in water and dilute to 250 ml. Chill 50 ml of this solution to 0°C, add successively 50 ml of 0.1N iodine solution and 20 ml of chilled 10% sodium hydroxide solution and allow to stand at 0°C for 90120 min. After adding 30 ml of 10% sulfuric acid, titrate with 0.1N sodium thiosulfate solution (factor, f) (consumed volume of the thiosulfate solution, a ml). Carry out the blank test in the same manner (consumed volume of the thiosulfatesolution, b ml). Calculate the content of pyruvate by the following equation.
Content of pyruvate (%)
=(b - a) ×f×k/ W × 500 × 1.011
Where k is the weight (mg) of pyruvate corresponding to 1 mlof 0.1N iodine solution (1.834 in sodium pyruvate, 1.867 in lithium pyruvate monohydrate).
On the other hand, commercial sodium pyruvate was purified to 99.7% (determined by the above method), which was used as the standard substance in colorimetric method for the quantitative determination of pyruvate with 2, 4-D. The pyruvate content of a sample was obtained by comparing the absorbance of the sample with that of 0.4 mM pyruvate standard solution.
