Luminol chemiluminescence reaction: Optimization by image analysis method and use in distinguishing human hemoglobin from potassium hexacyanoferrate(III) by addition of ascorbic acid

Abstract The luminol chemiluminescence reaction (luminol reaction hereinafter) is used to detect trace amounts of blood at a crime scene. In this study, luminol chemiluminescence was recorded with a smartphone and image analysis was performed to optimize the luminol reaction for use in distinguishing chemiluminescence produced by whole blood from that by iron(III) ions. The video file was analyzed by the image analysis software ImageJ and a two-dimensional chemiluminescence intensity vs. time graph was created. When luminol reagent that is composed of a mixture of 0.4%(w/v) luminol dissolved in 1.0%(w/v) sodium hydroxide aqueous solution and 1.0%(w/v) hydrogen peroxide aqueous solution (6:1) was used, chemiluminescence duration was increased in whole blood sample, whereas that in potassium hexacyanoferrate(III) sample was extremely short, namely, a spike-like peak was generated. Furthermore, when ascorbic acid was added to potassium hexacyanoferrate(III) in the stoichiometric ratio of 0.58 mol or higher to 1.0 mol of iron(III) ion, the false-positive result produced by iron(III) ions was suppressed. From the above results, by optimizing the composition of luminol reagent, it became possible to distinguish chemiluminescence due to whole blood from that due to potassium hexacyanoferrate(III) on the basis of the difference in chemiluminescence pattern. It was also suggested that the addition of ascorbic acid could eliminate false-positive results produced by potassium hexacyanoferrate(III).