A Simple Spectrophotometric Method for Determination of Glyoxylic Acid in Its Synthesis Mixture

Abstract

A new simple and reliable spectrophotometric method is described to determine glyoxylic acid in its synthesis reaction mixture containing oxalic acid, glycolic acid, acetic acid, glyoxal, and ethylene glycol by means of a modified Hopkins–Cole reaction between glyoxylic acid and tryptophan in presence of ferric chloride and concentrated sulphuric acid. The linear range of glyoxylic acid concentration is 0–0.028 M. The limits of detection (LOD) and quantitation (LOQ) are 0.0019 M and 0.00577 M, respectively. The LOD, LOQ, standard deviation, relative standard deviation, and recovery ratio of the proposed method are comparable with a selected HPLC reference method. Both methods displayed same precision and credibility. Reaction stoichiometry between tryptophan and glyoxylic acid is assumed to be 2 : 3. Reaction mechanism has been postulated based on identified molar ratios of reactants. Glyoxal gave a negative test with tryptophan although it is a dialdehyde.

A spectrophotometric method for the determination of tryptophan following oxidation by the addition of sodium hypochlorite pentahydrate

“…In addition, the Trp solution with a mixture of 19 standard amino acids, instead of glyoxylic acid, exhibited a brilliant crimson color change following the addition of NaOCl·5H 2 O ( S1A Fig ), and highly concentrated Trp solutions also showed the same color development without glyoxylic acid ( S1B Fig ). Although conventional Trp coloring methods, such as the Hopkins-Cole reaction [ 13 , 14 ], directly add the aldehyde reagent to the Trp solution, they have low reproducibility because of the instability of aldehydes and the side reactions caused by excess aldehyde reagent [ 23 , 24 ]. To overcome these problems, Kibrick adopted a method using methyl alcohol as an aldehyde source and observed a stable color reaction by gradually oxidizing methyl alcohol to formaldehyde and suppressing the generation of unintended byproducts in the reaction solution [ 23 ].…”

“…The maximum color development was obtained at 10°C, and the color change was diminished by an increase in temperature ( Fig 4B ). At a high temperature, the reaction products obtained by the condensation of Trp with aldehyde compounds may not be homogeneous and these byproducts can increase with the temperature [ 24 , 27 ]. Overall, the temperature and pH influence the reaction.…”

“…This result suggests that free Trp contributes to the coloring reaction and Trp-constituting protein is not involved. Moreover, glyoxylic acid relating to the Hopkins-Cole reaction is often included in cosmetics, food flavors, and medicine [ 24 ], but its presence (up to 200 mg/L) in the Trp solution did not affect the Trp absorption peak ( Fig 5D ).…”

“…To improve the Trp colorimetric reaction in the past, different reagents and conditions have been investigated, and various colorimetric methods have been developed [ 14 , 15 , 18 ]. Recently, Abdulwahed et al developed an improved Hopkins-Cole reaction for the simple and reliable chromogenic reaction of glycolic acid and tryptophan in the presence of ferric chloride and concentrated sulfuric acid [ 24 ]. Huang et al developed a simple, rapid, and highly selective Trp colorimetric method by improving the oxidation reaction of Trp and formic acid, an aldehyde compound, under acidic conditions [ 35 ].…”