EPA Method 327.0-REV1.1

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EPA Method 327.0-REV1.1:
Chlorine Dioxide and Chlorite in Drinking Water by Visible Spectrophotometry. Official Name: Determination of Chlorine Dioxide and Chlorite Ion in Drinking Water Using Lissamine Green B and Horseradish Peroxidase With Detection by Visible Spectrophotometry

A drinking water sample is collected headspace-free in a 16-mL amber glass vial. A second sample is collected and sparged with an inert gas to remove all traces of chlorine dioxide and then transferred to a second 16-mL amber vial. A third vial is filled with reagent water. A 1.0-mL aliquot of water is removed from each vial, and a 1.0-mL aliquot of a concentrated citric acid buffer containing glycine is added. The samples are capped and gently mixed, and then a second 1.0-mL aliquot is removed and a 1.0-mL aliquot of a Lissamine Green B (LGB)/Horseradish Peroxidase (HRP) reagent is added. The HRP catalyzes the conversion of chlorite to chlorine dioxide.(2,3) Chlorine dioxide rapidly oxidizes the LGB, reducing its absorption in the red region of the visible spectrum in proportion to the chlorine dioxide concentration.(4,5) A visible spectrophotometer is used to measure the absorbance of the reagent water blank and sample absorbance at 633 nm, which is the absorbance maximum for LGB in the citric acid/glycine buffer. The absorbance difference between the reagent water blank and the samples is used to calculate the concentrations of chlorine dioxide, using an external standard calibration curve, determined using chlorite standards. The unsparged sample is used to determine the total chlorite and chlorine dioxide concentration and the sparged sample is used to determine the chlorite concentration. The chlorine dioxide concentration is the difference between these two values.

This is a spectrophotometric method for the analysis of chlorine dioxide and chlorite* in finished drinking waters. It is primarily intended to be used at drinking water utilities in conjunction with daily monitoring requirements.

Datilio, T.A., and others, 2005, Method 327.0: Determination of Chlorine Dioxide and Chlorite Ion in Drinking Water Using Lissamine Green B and Horseradish Peroxidase with Detection by Visible Spectrophotometry, EPA 815-R-05-008.

Free available chlorine reacts with LGB to discolor the solution. Glycine reacts with the free available chlorine to form chloroaminoacetic acid, thereby eliminating the potential for this type of interference. Laboratory glassware can potentially exhibit a demand for chlorine dioxide. While this was not observed during method development, it could bias the analytical results and lead to poor accuracy and/or precision. This potential problem can be avoided if sample collection vials are cleaned and reused as described in Section 8.1.2 of the method. Chlorite concentrations are calculated after the samples are sparged to remove chlorine dioxide. Samples that are not efficiently sparged to remove the chlorine dioxide will yield chlorite values that are higher than the true value.

QC Requirements:
Initial demonstration of accuracy (which must be + or - 30% of fortified value), precision (RSD must be

Maximum Holding Time:
4 hours



0.2 - 2.2 mg/L


Detection limits were determined using a procedure similar to the Method Detection Limit (MDL) procedure at 40 CFR 136, Appendix B. Detection limits (DLs) were determined by analyzing seven replicates of fortified reagent water containing only the target analyte (i.e., only chlorite or only chlorine dixoide); see Table 1. Additional data, including DLs with both analytes present, are available in the method.

Revision Number:
Revision 1.1, May 2005