Environmental Laboratory Exercise
Analysis of Hydrogen Peroxide by Fluorescence Spectroscopy
Judith Weinstein-Lloyd
Chemistry/Physics Program, SUNYICollege at Old Westbuly, Old Westbuly, NY 11568
Jai H. Lee
Environmental Chemistry Division, Brookhaven National Laboratory, Upton, NY 11973
Gaseous hydrogen peroxide is found in parts-per-billion
by volume concentrations in clean and polluted atmos-
pheres. Studies show that HzOz and organic peroxides are
deleterious to plant tissues (I, 2), and may play a part in
forest decline in Europe (3).A number of analytical meth-
have heen developed for determining atmospheric per-
oxides; these methods and their applications in the field
are the subject of two recent reviews (4,5).
~ ~ofhighiconcentrations
~ ~
ofozone,dhydroear~ons,
~ ~
particulate matter and oxides of that occur in ur.
ban areas during the summer, sometimes referred to a s
'cLosAngeles smog", are complex chemical events initiated
by sunlight and involving the free radicals OH* and HOz*
a s intermediates. Figure 1illustrates the key chemical re-
actions involved in the production of photochemical air pol-
lution. In spite ofthe central role played by free radicals,
low ambient concentration makes their measurement in
the field diff~cult.One of the ways we verify our under-
standing of photochemical air pollution is by comparing
field measurements to the results of computer models (e.g.,
6,7) that predict the time evolution of key chemical species
under specified meteorological conditions. Products such
Nlrate
aemwl
Deposition
Sulfate
aerosol
',
Figure 1. Principal reactions of gas-phase species implicated in the formation of acid rain and
photochemical pollution.
a s 03, NOZ, HN03, and HzOz are easier to measure than
their highly reactive free-radical precursors. Such meas-
urements in conjunction with model calculations provide
information about free radical concentrations.
The effect of acidic precipitation on streams and lakes in
the Northeastern United States and Canada was well-
documented during the 1980's (8, 9), as were the conse-
quences to building materials and the ecosystem (9-11). It
has been demonstrated that precipitation in these areas
contains sulfuric acid, the oxidation product of SOz gener-
ated during the combustion of sulfur-rich coal. The princi-
pal reactions involved in acid rain formation are illus-
trated in Figure 1.Although gas-phase oxidation of SOz by
OH. radical to acid formation, aqueous.phase
oxidation pathways are believed to he more important, as
illustrated by the increase sOzoxidation
rates in power plant plumes that impact fog banks (12,13).
principal aqueous-phase oxidants for soz are H ~ o ~03, ,
and, in the presence of transition metal ions, oz,with
HzOz playing the most important role in the Northeast
(14). Its large Henrys Law coeff~cient,7.4 x lo4 MIatm,
ensures substantial partitioning of Hz02 into the aqueous
phase (15). Field studies have demonstrated that cloud
droplets typically contain either dis-
solved SOz or HzOz, but not both, sug-
gesting t h a t t h e reaction between
them goes to completion during a typi-
cal cloud lifetime (161, and laboratory
studies have elucidated the mecha-
nism of t h e H20z-S(IV) reaction,
which exhibits pH-dependent kinetics
(17,181. Because HzOz may be the lim-
iting reagent in the vicinity of SOz
emitters, predicting the extent of SOz
oxidation requires accurate determi-
nation of Hz02 in the atmosphere (19).
Recent evidence has emerged suggest-
ing that organic peroxides are present
in the atmosphere and also play a role
in S(IV) oxidation (20,211.
I n this e x ~ e r i m e n t .students ana-
lyze precipitation samples for trace
concentrations of Hz02 using a newly
developed fluorescence technique (22).
/ I Analysis is based on the ~roductionof
OH.-radical by the reaction between
HzOz and ferrous ion (Fenton's re-
agent) with subsequent radical scav-
Dewitiw enging by benzoic acid:
Fe(I1)+ H,O, + OH. + OH- + Fe(II1)
(1)
(2)
OH. + C,H,COOH + C,H,(OH)COOH.
Volume 72 Number 11 November 1995 1053
 I n the p n w n c e of02 and dissolved iron, the cyclohrxadi-
myl radical formed in re;ictlon 2 produces fluorescent hy-
drox\.benzox
~~-~~-" ~ ~ acid
~ - - OIIBAI
~ ~ isomers 12.71. The substantial
increase i n fluoreicence intensity of OHBA upon com-
nlexation with Al(II1) ion is ex~loitedto increase sensitiv-
ity (24). Unlike standard sp~ctroscopicand titrimetric
techniques, Fenton-OHBAanalysis is sensitive enough for
the determination of ambient peroxide i n precipitation,
which typically varies from to 104M. And, in contrast
to a more widely used, highly sensitive enzyme-based
method (251, the reagents are inexpensive and easily avail-
able, and reagent solutions are stable, making this experi-
ment well-suited for the undergraduate laboratory.
Experimental procedure'
Ramwater samplei; are collected a s closc to the time of
the srheduled (!xperiment as posiible, and pH measured.
To account for matrix effects, samples are spiked with
small nliquots ofa known hydrogen peroxide standard and
a~ n~n l"-v ~
- e
-~dbv
~"
~~-ronventional
~ ~ standard additions methods
(26). For good precision, the instructor should make a pre-
liminary determination of the Hz02 concentration and use
a peroxide standard which, when diluted, will approxi-
mate the unknown concentration. Although HzOz can be
purchased in assayed 3% solution from Mallinkrodt (St.
Louis. MO). the concentration eraduallv declines, so i t is
advisable to titrate the 3% solition against standardized
KMn04. We have found that HzOz keeps its titer for sev-
eral months when refrigerated and protected from con-
tamination. If rainwater collection is impractical, a n HzOz
unknown can be prepared by the instructor.
A multichannel peristaltic pump is used to deliver the
product of reaction between simple, fluorescence reagent
(iron(I1) sulfate and benzoic acid) and enhancing reagent
(Al(II1) i n acetate buffer) to a fluorometer flow-through
cell, as illustrated in Figure 2. The reaction coils i n the
figure are lengths of Teflon tubing that introduce a two-
minute delay to allow the reactions to proceed substan-
tially toward completion. If a pump i s unavailable, equal
volumes of the sample and fluorescence reagent can be
mixed, and a third equal aliquot of enhancing reagent
'Adetaiied experimental procedure is available from J. W: L. upon
request.
RECORDER FLUOROMETER
ENHANCING
REAGENT
FLUORESCENCE
REAGENT - PUMP
PEROXIDE
WPLE
-
Figure 2. Experimental setup for H202 measurement. Fluorescence
reagent contains 0.8 mM FeSO, and 2.0 mM benzoic acid in pH I .9
Sulfuricacid; enhancing reagent contains 6.0 mM AI(NO,), in 0.10 M
acetate buffer, pH 3.8. Peroxide sample preparation is described in
text.
1054 Journal of Chemical Education
added. The reaction with fluorescence reagent should be
timed for three minutes; the AI(II1) complexation will be
complete in 1 5 min. Maximum fluorescence intensity of
the hydroxybenzoic acid-aluminum complex under t h e ex-
perimental conditions described above is obtained with
hex=305 and L,=410 nm (10).
The low peroxide concentrations determined in this ex-
periment call for careful analytical work and attention to
detail. Afew precautions are noted below
Many common impurities,including detergents, fluoresce. It
is imoartant to clean and rinse thorouehlv all dassware that
will came into contact with peroxide samples. Rinsing with
nitric or hydrofluoric acid may be necessary to remove traces
of organic impurities.
Background fluorescence can be minimized by using freshly
prepared reagents that have not absorbed HzOzfrom the air,
and protecting solutions from light. Students should be cau-
tioned to prepare all reagents from the same water for con-
sistent results.
Rainwater should not have im~actedtrees or other obiects
are minimized.
Safety Considerations
Safety glasses and protective clothing should he worn for the
proced;re.
Concentrated sulfuric and acetic acids, required far reagent
preparation, must he used with caution.
.Cancentrated nitric or hvdrafluorie acid. which mav be
Results
A typical graph of fluorescence intensity versus volume
of added standard HzOz is shown in Figure 3. For this rain
sample, the concentration of HzOz, determined from the
ratio of intercept to slope, a s described in this Journal (261,
is 1.2 x 10" M. On rare occasions, rain samples show no
measuratde H202 because it has heen rirrim>dby :in t!~c(~.;s
of dissolved S02. When this occurs, cahbrauon cJrves may
exhibit nonlinearity due to reaction between SOz a n d
added HzOz. I t should be noted that the Fenton-OHBA
method shows limited sensitivity to some organic perox-
mL H202 ADDED
Figure 3. Results of standard addition analysis of H 0 in recipita-
2 3M) added to
tion. Abscissa shows milliliters standard H202 (4.5x 10-
25 mL samples

ides in addition to HzOz. However, its high solubility and
greater stability under ambient conditions make HzOz the
predominant peroxide observed in precipitation.
Conclusion
This experiment is appropriate for an upper-level instru-
mental analysis or integrated laboratory course. Although
i t can be conducted a s a s i m.~ l auantitative
e. Drocedure us-
ing commercial HzOz unknowns, the experience can be
-
broadened bv havine students collect and handle authen-
tic environmental samples, examine the excitation and
emission s ~ e c t r u mof the fluorescent product, or analyze
8. "Interim Assessment: The Causes and Effects of Aeid Deposition:' National Acid
F'mipitationhsessment Prprpam. Government Printing Office: Washmgton. DC,
1987.
9. Hicks. B. B.. Ed. Drpoailion. Both We1 ond Dry: Aeid Precipitation Series Volume 4,
Butteworth: MA. 198C
lo. Tomanson, G. H E N a f f f A d d Depmi,ii" in I h h F " ~ s t 1 " r E " m p ~ ~ " d N ~ ~ t h A m e ~ .
i m : Lerner Publications: MN. 1990.
11. Allen. R. 0.:Allen. C. R.: Sherwood. S. Today's Chem~afo i Work 1993.2.28-38.
12. Eat0ugh.D. J;Arthurn.R. L.;Etough, N. L.; Hill. M. W.;Mangelsan. N. E:Richter,
B. E.: Hansen, L. DEnviron. Sei. Technol. 1984.18.855-859.
13. Bizjak,M.;Benner W.H.:Hanren.A.D.A.:Hrcek.D.:Hudnik,Y:Novakov,TAtmos.
Enuimn. 1988,22.2861-2862.
14. Schwartz. S. E. In Sol, NO and NO2 Ozidorion Mpehonisms:Atmospherk Consid-
erations; Calvert. J . G., Ed.. Butterworth: MA. 1984.
15. Lind. J. A : Kok, G. L. J. Oeoph.ys. ROS.1886.91.7889-7895.
16. Daum. P. H.: Schwartr, S. E.; Kellx T J.: Newman. L. Alrnos. Enuimn. 1984. 18.
*<", L--9CS"
---.

11. Martin. L. R.:Damschen. D . E A t m o r Enuimn. 1981.15. 1615-1621.

samples for S ( N ) and other inorganic ions. 18. Lee,Y -N.:Shen. J.;Klotz, P. J.; Schwartz. S. E.;Ncwman,L.J. GPophyaRes. 1986,
Blank solutions may exhibit substantial background 91.1326P13274.
fluorescence, t h e use of the most sensitive 19. Kleinman,L. 1;Daum.P. HAfmas. Enuiron. 1991,25A, 2023-2028.
20. Hellpointner E.; Gab. S. Noturr 1989.337. 631-634. 21. Zhou, X.; Lee. Y. - N J.
scales on some fluorescence detectors. This, coupled with Phys Chsrn. 1992,96,265-272.
low :lmlicmt peroxide conrentratitmi may challenge in- 22. Lee. J. A.;Tang. 1. N.; Weinstein-Lloyd. J . B.Anol. Chern. 1990.62.2381-2381.
23. Oorfman, L. M.;Tauh, I. A.;Buhler, R. E. J. Chem. Phys 1962,36, 30515061.
strument detertion limits in this experiment Unlike mo?t 24. Lee. J . H.: Tang. 1. N.; Weinstoin-Lloyd. J. B.: Halpel E. B. Enuiron. Sci. T-hnol.
ronventionnl laboratory exercises that specificall? are de- 1994.28, 118h1185.
signed to avoid such conditions, this rxp,!rimt!nt gn.es stu- 25. Lazrus.A. L.: Kok,G. L.:Gitlin, S.: Lind. J. A A n o ! Chom. 1985.57.917-922.
26. Bsder,M. J Chern. Educ 1980,57.703-706.
denti wluahls prnctlcal experience with sonic of the other- 27. Lee, J. H.; Leahy, D. P.:Tmg. 1. N.: Newman, L. J Oeophya. Res. 1998.98, 2911-
wile iibstract concepts ule teach; i.e, nose, detection limit 2915.
;ind scllsitivlty. Rerause hydrogen peroxide is ubiquitous 28. Weinstein-Lloyd, J. 8 . : Daum, E H.; Nunnemacksi L. J.; Lee. J . H.; Kleinman, L.
I. J G~ophyn.ROS.1995, in press.
in the atmosphere and highly soluble, the experiment also 29. Schwsriz, S. E. Science 1989,243,753-768.
illustrates tht. critical imDonance o f hlank mrnsurernents.
The Fenton method has been used recently to monitor
peroxide concentrations i n air, cloudwater, precipitation
and surface waters in a number of field missions (27,281.
Our students have carried out this experiment for several
years with good results. They are enthusiastic about col-
lecting and analyzing authentic precipitation samples.
The experiment also serves a s a point of departure for dis-
cussions on environmental chemistry and environmental
regulation. The 1990 Clean Air Act mandated a 10-million
ton per year reduction in SOz emissions from 1980 levels
through improved scrubber technology and the use of SOz
emissions trading allowances, a market-based approach
toward environmental imnrovement. Each emitter is Dro-
vided with a number of emission allowances that can be
used to emit SOz or can be sold to other emitters by plants
that successfully reduce their own emissions. Both HzOz
and SOz are required for the production of acid rain, as
illustrated i n Figure 1, but peroxide must be present i n
excess if the mandated SOz reductions are to have the de-
sired effect. Chemistry students who understand the con-
c e ~oft the limiting reagent know that the total amount of
(e.g., ~ 2 ~ 6 cannot
4 ) be decreased by lowering re-
actant concentration (e.g., SOz) if HzOz is a limiting re-
agent. Details such as this illustrate the role of careful sci-
entific work in formulating public policy (29).
Acknowledgment
We are grateful to E. Garrity, L. Newman, S. Schwartz,
and I. Tang for many helpful discussions. This work was
supported by NSF-RUI grant ATM-9112698 and DOE
grant DE-FG02-91ER61206 to J. W.-L and conducted un-
der Contact No. DE-AC02-76CH00016 with the U.S. De-
partment of Energy under the Atmospheric Chemistry
Program within the Off~ceof Health and Environmental
Research.
Literature Cited
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2. Sohel, F HNotarr 1956, 177.977-983.
3. Masuch. G.: Kettrup. A.: Mallant, R. K. A. M.; Slanina. J . In,. J. Envimn. Anal.
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.-""-.-"".
4. Sakugswa, H.; Kaplan. I. R.: Tsai. W.: Cohen, Y.; Enuiron. Sci. lkhnol. 1990, 24.
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3°K-

5. Gunz, D.: Hoffman, M. RAtrnm. Enuiron. 1390.24.1601-1633.

6. Jacob. D.J.;Oottlieh. E. W.: Rsther,M. J.: J. C m p h y 8 . R ~ ~
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7. Chang,J. S.;Bmst.R.A.;lsskseu. S.A.: Madronich, S.;Middleton,P.;Stockwell,W.
R.: Wa1eek.C. J. J. Oeophyr Re8 1987.92. 14681-14700.

Volume 72 Number 11 November 1995 1055