Environmental Engineering Reference
In-Depth Information
The flow lines were made of Teflon tubing (i.d.
=
0.5 mm). After filling up with
carrier ultrapure water and 0.6 M NaOH solution, all flow lines should be freed
from air bubbles before starting. The fluorescence detector should be set at Ex/
Em
=
320/400 nm, and the zero level of fluorescence recorded. After completion
of the baseline one should set again the fluorescence level to zero, then the analysis
can be started. After completion of the measurements, before turning off the plunger
pump, one should wash the flow lines. In particular, the NaOH line should be
flushed with water and the outgoing flow should be checked for pH until neutrality.
In sample preparation, 1 mL sample in a Teflon or glass container is first
treated with catalase (20
μ
L, 500 units mL
−
1
) in order to decompose all the H
2
O
2
present (Eq.
2.2
), shaking well for a few seconds and keeping still for six min-
utes. This solution can be used as a blank. Moreover, 1 mL of the same sample
where catalase is replaced with 20
μ
L of ultrapure water is used to obtain the sig-
nal from H
2
O
2
. Fluorescence can be induced upon addition (300
μ
L) of peroxi-
dase mixed with
p
-hydroxyphenylacetic acid. The difference in the fluorescence
values (Ex/Em
=
320/400 nm) between samples treated with catalase and those
without the enzyme will provide the estimate of H
2
O
2
concentration. Calibration
can be carried out by use of the external standards already described (Fig.
8
a).
A typical example of calibration curves for standard H
2
O
2
and peracetic acid
Fig. 8
A typical example of
calibration curve for aqueous
solutions of standards
H
2
O
2
(
a
) and peracetic acid
(CH
3
OOOH) (
b
) measured
using this fluorometric
method
200000
(a)
160000
120000
80000
y = 33.4760x + 17372.1980
R
2
= 0.9999
40000
0
0
1000
2000
3000
4000
5000
Standard H
2
O
2
(nM)
25000
(b)
20000
15000
10000
y = 30.2738x + 9410.5676
R
2
= 0.9990
5000
0
0
100
200
300
400
500
Standard CH
3
OOOH (nM)
