Microwave photochemical reactor for the online oxidative decomposition of p-hydroxymercurybenzoate (pHMB)-tagged proteins and their determination by cold vapor generation-atomic fluorescence detection

Anal Chem. 2013 Dec 17;85(24):12152-7. doi: 10.1021/ac403389z. Epub 2013 Dec 2.


A novel method is presented for the characterization and determination of thiolic proteins. After the labeling with p-hydroxymercurybenzoate, the pHMB-labeled proteins underwent on-line oxidation with a novel microwave (MW)/UV photochemical reactor, followed by cold vapor generation-atomic fluorescence spectrometry (CVG-AFS) detection. The MW/UV process led to the conversion of pHMB to Hg(II) with a yield of 89.0 ± 0.5% without using chemical oxidizing reagents and avoiding the use of toxic carcinogenic compounds. Hg(II) was reduced to Hg(0) in a knotted reaction coil with NaBH4 solution, stripped from the solution by an argon flow and detected. The chromatographic method for labeled thiolic peptides was linear in the 0.2-100 μmol L(-1) range, with a LOD as mercury of 57 nmol L(-1). This system has proven to be a useful interface for liquid chromatography coupled with CVG-AFS in the determination and characterization of thiolic proteins. This method has been applied to the determination of thiolic peptides after tryptic digestion of serum albumins from different species (human, bovine, rat, horse, and sheep).

MeSH terms

  • Animals
  • Cattle
  • Chromatography, Reverse-Phase
  • Cold Temperature*
  • Flow Injection Analysis
  • Humans
  • Hydroxymercuribenzoates / chemistry*
  • Microwaves*
  • Oxidation-Reduction
  • Photochemical Processes*
  • Proteins / analysis*
  • Proteins / chemistry*
  • Proteins / metabolism
  • Rats
  • Spectrometry, Fluorescence / methods*
  • Trypsin / metabolism
  • Ultraviolet Rays
  • Volatilization


  • Hydroxymercuribenzoates
  • Proteins
  • 4-hydroxymercuribenzoate
  • Trypsin