Optimization of post-column chemiluminescent detection for low-molecular-mass conjugates of acridinium esters

John G. Rollag, Tsentao Liu, David S. Hage

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


In this study, various factors are examined that affect the post-column chemiluminescence detection of low-molecular-mass compounds labelled with acridinium esters, using 9-phenyl acridinium eater (PAE) as the initial model. Reaction conditions examined included the effect of hydroxide or hydrogen peroxide concentration, ionic strength and surfactant content on the degree and length of light production by the acridinium ester label. Based on these results, a system was developed for the post-column detection of acridinium ester conjugates in HPLC. The final post-column reactor had optimum conditions similar to those for a benchtop luminometer but gave light production over slightly longer periods of time and had a broader range of reagent concentrations that gave a maximum response. The detection limit of this system for PAE was 7 x 10-19 mol per injection, with linear and dynamic ranges that extended up to 3 x 10-15 and 3 x 10-13 mol, respectively. Some preliminary work was conducted examining the use of this system in chromatography-based competitive binding immunoassays with a thyroxine-acridinium ester conjugate being used as the label. The estimated limit of detection was 2.5 x 10-17 mol (or roughly 10-12 M for a 25 μl sample) based on the retained fraction of the conjugate.

Original languageEnglish (US)
Pages (from-to)145-155
Number of pages11
JournalJournal of Chromatography A
Issue number2
StatePublished - Mar 28 1997


  • Acridinium esters
  • Chemiluminescence detection
  • Detection
  • LC
  • Thyroxine

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry


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