Analysis of the Morgan-Elson chromogens by high-performance liquid chromatography

Academic Article

Abstract

  • The Morgan-Elson method for quantitative N-acetylhexosamine analysis is a two-step procedure comprising alkali treatment of the sugar and subsequent condensation of the resulting chromogens with p-dimethylaminobenzaldehyde (Ehrlich's reagent) to yield a colored product. In the present investigation, the products formed in the first step of the procedure were analyzed by high- performance liquid chromatography (HPLC) on a reversed-phase (C18) column, which was eluted with a water-methanol gradient; the absorbance of the effluent was monitored at 229 nm. The profile generated from alkali-treated N-acetylglucosamine exhibited two major peaks, in a ratio of ~2.5:1, which accounted for 94% of the total peak area. A third peak, accounting for 3% of the peak area, was eluted in an intermediate position, and several smaller peaks were also observed. The three predominant components, isolated by preparative HPLC, all gave a purple color on addition of Ehrlich's reagent, indicating that they were Morgan-Elson chromogens. The HPLC profile of alkali-treated N-acetylmannosamine was identical to that of the products generated from N-acetylglucosamine, as was expected because of the elimination of the asymmetry at C-2 during formation of the chromogens. N- Acetylgalactosamine yielded two major peaks, which were eluted in the same positions as the two major products formed from N-acetylglucosamine, but the intermediate peak seen in the N-acetylglucosamine pattern was absent. The HPLC procedure allowed detection of as little as ~ 25 ng of N- acetylglucosamine and may therefore be of value as an alternative to the complete Morgan-Elson procedure when only small amounts of sample are available for quantitative analysis.
  • Authors

    Digital Object Identifier (doi)

    Author List

  • Rodén L; Yu H; Jin J; Ekborg G; Estock A; Krishna NR; Livant P
  • Start Page

  • 240
  • End Page

  • 248
  • Volume

  • 254
  • Issue

  • 2