Optimization of a high-performance liquid chromatography method to quantify bilirubin and separate it from its photoproducts: Effect of column length, pH, mobile phase composition, and flow rate

A. M H Al-Hamdi, J. R. Williams, S. M Z Al-Kindy, A. E. Pillay

Research output: Contribution to journalArticle

11 Citations (Scopus)


A rapid reversed-phase (RP) high-performance liquid chromatography method for the isolation of bilirubin from its photoproducts (e.g., biliverdin) is reported. The method is based on isocratic elution using methanol:water as the mobile phase. A 24 full-factorial experimental design approach was adopted. For the optimization, the best separation was obtained using a flow rate of 1.50 mL/min, a mobile phase of 99:1 methanol:water (v/v) at pH 3.60, and a 150 × 4.6 mm id RP (C18) column containing 5-μm particles. These conditions produced the fastest total retention time of 3.38 ± 0.055 min, and other chromatographic parameters were acceptable. Under the optimum conditions, a linear calibration curve for bilirubin was obtained over the 1.0-40.0 μg/L concentration range studied. The limit of quantification was 0.79 g/L and the limit of detection was 0.24 μg/L. Bilirubin in solution was monitored by ultraviolet detection at 450 nm.

Original languageEnglish
Pages (from-to)209-218
Number of pages10
JournalApplied Biochemistry and Biotechnology
Issue number3
Publication statusPublished - Dec 2006



  • Bilirubin
  • Biliverdin
  • Experimental design
  • Isocratic elution
  • Reversed-phase chromatography

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Bioengineering

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