Determination of L-ascorbic acid in tomato by isotachoelectrophoresis. Application of computer simulation system for setting of determination conditions to avoid the mixed-zone problem.

Abstract

When the isotachoelectrophoretical method is used with HCl-beta-alanine as the leading electrolyte solution (pH 3.6), ascorbic acid forms a mixed zone with glutamic acid, and it is difficult to separate the two. Therefore, the application of a computer simulation system to separate the two acids was investigated. This system can be used with compounds for which the absolute electrophoretical mobility and dissociation constant have already been determined. Values for glutamic acid alone were entered in the data bank "SIPS-1", and then those for ascorbic acid were estimated from the values of similar compounds. Values for aspartic acid were used, and the electrophoretical behavior of ascorbic and glutamic acids was simulated at pH range from 3 to 9.9. Based on this simulation, separation appeared possible at pH 9.0 to 10.0, and indeed, experimental determination of ascorbic acid was successfully performed with a leading electrolyte solution composed of HCl-ethanolamine (pH 9.4) without any glutamic acid interference. Even though ascorbic acid is highly susceptible to oxidation in an alkaline medium, because the ascorbic acid solution is injected at the interface of the leading and terminal electrolyte solutions in the electrophoretical procedure, it does not come in contact with air during electrophoresis. This explains why ascorbic acid can be determined in an alkaline medium.