Environmental Engineering Reference
In-Depth Information
2 Fluorometric Method for Determining H 2 O 2 and ROOH
in Natural Waters
Theory : The fluorometric method described here has been developed by
Fujiwara et al. ( 1993 ) and Sakugawa et al. ( 2000 ) by implementation of ear-
lier methods (Lazrus et al. 1986 ; Guilbault et al. 1968 ; Miller and Kester
1988 ). The compounds H′OOH (where H′ = H or CH 3 -, -OCH 3 , etc.) react
with p-hydroxyphenyl acetic acid in the presence of peroxidase, to produce the
6,6′-dihydroxy-3,3′-biphenyldiacetic acid (POPHA dimer: Eq. 2.1 ). The latter is
detected using a fluorometer at excitation/emission = 320/400 nm.
CH 2 COOH
CH 2 COOH CH 2 COOH
peroxidase
(2.1)
2
+
H 2 O 2
OH
OH
OH
2H 2 O 2 catalase
(2.2)
−→ 2H 2 O + O 2
A sodium hydroxide solution is used to increase the pH to approximately 12,
which largely enhances the fluorescence intensity of the POPHA dimer. In this way
it is possible to detect few nano molar (nM) levels of H 2 O 2 in natural waters. To
make the analytical blanks and to distinguish H 2 O 2 from ROOH, one should add
catalase to the samples, which causes the rapid decomposition of H 2 O 2 (Eq. 2.2 ).
2.1 Chemicals Preparation
Note: ultrapure water should be used throughout. It should be kept in the dark for
3 days before use to allow for the decomposition to undetectable levels of H 2 O 2
and ROOH, which could possibly be present.
Preparation of p - hydroxyphenyl acetic acid solution :
(i) Take potassium hydrogen phthalate (71.48 g) in approximately 650 mL water
in a 1-L beaker, and dissolve it at approximately 40 °C under gentle stirring.
(ii) Dissolve 12 g NaOH in approximately 50 mL water in a 100 mL beaker.
(iii) The pH of the solution (i) is adjusted to 5.5 upon addition of solution
(iii) under constant stirring.
(iv) Add 18.62 g of di-sodium dihydrogen ethylenediamine tetraacetate dehydrate
(EDTA) to the solution (iii) under constant stirring. The EDTA is added to
eliminate the effect of metal ions, particularly Fe 2 + , and to prevent the for-
mation of a Mg(OH) 2 precipitate after addition of NaOH to seawater samples
(Fujiwara et al. 1993 ). It can be noted that without EDTA, 1 mg/mL Fe 2 + can
reduce the signal intensity by 80 % (Fujiwara et al. 1993 ).
(v) Add 0.304 g of p -hydroxyphenyl acetic acid to the solution (iv) under con-
stant stirring, then adjust the total solution to 1-L in a volumetric flask.
 
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