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Determination of hydrogen peroxide by micro‐flow injection‐chemiluminescence using a coupled flow cell reactor chemiluminometer
Author(s) -
Nozaki Osamu,
Kawamoto Hiroko
Publication year - 2000
Publication title -
luminescence
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.428
H-Index - 45
eISSN - 1522-7243
pISSN - 1522-7235
DOI - 10.1002/1522-7243(200005/06)15:3<137::aid-bio576>3.0.co;2-j
Subject(s) - luminol , chemiluminescence , hydrogen peroxide , chemistry , chromatography , reagent , volumetric flow rate , flow injection analysis , reproducibility , light intensity , analytical chemistry (journal) , detection limit , biochemistry , physics , optics , quantum mechanics
A novel flow cell reactor was developed for micro‐flow injection determination of hydrogen peroxide (H 2 O 2 ) using horseradish peroxide (HRP)‐catalysed luminol chemiluminescence. The newly developed flow cell reactor for a chemiluminometer allowed mixing of the chemiluminescent reagents in front of a photomultiplier for maximum detection of the emitted light. The rapid mixing allowed a decrease in the flow rate of the pump to 0.1–0.01 mL/min, resulting in increased sensitivity of detection of light. The flow cell reactor was made by packing HRP‐immobilized gels into a flow cell (Teflon tube; 6 cm × 0.98 mm i.d.) located in the cell holder of a chemiluminometer (flow‐through type). The HRP‐immobilized gels were made by immobilizing HRP onto the Chitopearl gel by the periodate method. H 2 O 2 specimens (50 µL) were injected into a stream of water delivered at a flow rate of 0.1 mL/min and mixed with a luminol solution (0.56 mmol/L in Tricine buffer, pH 9.2) delivered at 0.1 mL/min in the flow cell reactor. Within‐run reproducibility of the assay of H 2 O 2 was 2.4% (4.85 µmol/L; flow rate 0.1 mL/min, injection interval 10 min). The reproducibility of the H 2 O 2 assay was influenced by the flow rates and the injection intervals of the H 2 O 2 specimens. As the flow rates decreased, both the light intensity and the light duration increased. Optimal light intensity was obtained at a luminol concentration of 3–8 mmol/L, but 0.56 mmol/L was sufficient for assay of H 2 O 2 in clinical specimens. At a luminol concentration of 0.56 mmol/L, the regression equation of the standard curve for H 2 O 2 (0–9.7 µmol/L) was Y = 27.5 X 2 + 394 X + 58.9 (Y = light intensity; X = concentration of H 2 O 2 ) and the detection limit of H 2 O 2 was 0.2 µmol/L. This method was used to assay glucose (2.7–16.7 mmol/L) based on a glucose oxidase (20 U/mL, pH 7.4) reaction. The standard curve for glucose was Y = 167 X 2 − 351 X + 1484 (Y = light intensity; X = glucose). The within‐run reproducibility for an aqueous glucose standard (2.7 mmol/L) and a control serum (glucose, 5 mmol/L) was 4.48% ( n = 5) and 5.70% ( n = 9), respectively. © 2000 John Wiley & Sons, Ltd.