trends in analytical chemistry, vol. 17, no. 10, 1992

Hydrogen peroxide in the marine environment: cycling and methods of analysis

David Price, Paul J. Worsfold and R. Fauzi C. Mantoura Plymouth, UK

Hydrogen peroxide (H202) is a key intermedi- ate in many biological and environmental processes and there are a range of analytical methods available for its quantification, e.g. spectrophotometry, fluorimetry, chemilumi- nescence and electroanalytical techniques. This article focuses on the determination of H202 in seawater, wherein it is believed to play a significant role in redox reactions and the photochemical oxidation of dissolved organic matter. The cycling of H202 in seawater and potential approaches to in-situ monitoring are discussed, with particular reference to chemi- luminescence techniques.

Hydrogen peroxide in seawater

Hydrogen peroxide (H202) is ubiquitous in the hydrosphere and has been shown to be present in freshwaters [l], groundwaters [2] and estuarine [3] and marine [4] environments. Table 1 shows the range of reported concentrations in these matrices and a schematic diagram showing the cycling of Hz02 in seawater is shown in Fig. 1.

Hydrogen peroxide is believed to play an impor- tant role in redox reactions in the ocean, e.g. the “photo-Fenton reaction” involving iron( organic matter and hydroxyl radicals reported by Zepp et al.

[5]. It may also react directly with pollutants and a

TABLE 1. Natural levels of hydrogen peroxide in the hydro- sphere

Water Concentration

(nV

Ref.

Freshwater 50-3200 6 Seawater - surface 100-300 20 Seawater - deep c5 20 Rainwater 8000-80 000 13

016S-9936/92/$OS.O0

379

variety of naturally occurring dissolved and particu- late constituents [6]. Hydrogen peroxide has been used in wastewater treatment because it oxidizes living material and can therefore reduce the bacterial count in sewage effluent [7]. It is a reactive species and in seawater is produced, at least in part, from the disproportionation of the superoxide anion radical, 0~~‘which is believed to be generated by light in- duced electron transfer processes involving molecu- lar oxygen, dissolved organic chromophores and transition metal ions [5]. For example the general reaction scheme with humic substances (HS) [8] is:

HS+hv=HS*

HS* + 02 = HS+ + 02-’

The redox cycling of metals such as copper(I) with 02 has been suggested as a source of 02- [9]:

HETEROGENEOUS PHOTOCATALYSED

REDOX REACTIONS

CYCLING /

HO;- H,0,

/ \ ATMOSPHERIC BIOLOGICAL DEPOSITION PROCESSES

Fig. 1. H202 cycling in the ocean.

0 1992 Elsevier Science Publishers B.V. All rights reserved

380 trends in analytical chemistry, vol. 11, no. 10, 1992

Other metals such as chromium, iron and manga- nese have also been studied, suggesting that they may play a role in the generation of 02” [8]. Heterogene- ous photocatalysed reactions may result in the forma- tion of 02‘_: for example Oliver et al. [lo] found that titanium(W) oxide could give rise to 02-‘production in natural waters. The relative contributions of these processes are not known. 02-‘is unstable in aqueous solution and undergoes rapid disproportionation to Hz02 and 02 [ll]:

pathway is the major source of Hz02 to the ocean although the contributions from atmospheric deposi- tion and biological processes have not been quanti- fied [8]. - -

In seawater, as in all natural waters, the rate of formation of Hz02 cannot be quantified due to its simultaneous decomposition and therefore the term accumulation rate is employed [8]. Decomposition is carried out mainly by fast biological processes (via catalase and peroxidase activity) but slower photo- chemical oxidation has also been observed [ 161.

202-’ + 2H+ = Hz02 + 02

The reaction is not a simple one step mechanism but proceeds via a variety of pathways. The addition of superoxide dismutase catalyses this final reaction and this has been used to strongly link the involve- ment of 0~~‘in the formation of Hz02 [ 121. The same workers showed that 24-41% of the 02-• produced did not lead to the formation of H202, indicating that other mechanisms for consumption and/or destruc- tion may exist.

Diurnal variability in Hz02 concentration has con- firmed that photochemical processes are partly re- sponsible for its formation [ 1,4,16]. Cooper and Lean [l] monitored the level of Hz02 in surface lakewater and found that the concentration increased rapidly from less than 10 rUJ4 in the morning , to a peak of as much as 800 nM in the afternoon, before decreasing back to below 10 nh4 in the evening. The Hz02 concentration was also found to be very sensitive to the extent of the cloud cover.

Hydrogen peroxide is also supplied to the marine environment by wet and dry deposition of photo- chemically produced Hz02 in the atmosphere. Rain can contain relatively high levels of Hz02 but is not a significant source to the ocean, although transient increases have been observed during rain events [ 131.

The vertical distribution of Hz02 in the ocean is directly proportional to the amount of light received; highest levels are found in the photic zone and the levels decrease exponentially as the light is attenu- ated by scattering and absorption in the water column D71.

Finally, biological processes are known to produce Hz02 in the marine environment, for example, ex- tracellular production by cyanobacteria [ 141 and the marine phytoplankter, HymenOmOnas carterue [ 151 have been reported. H.carterae was found to produce a high level of Hz02 (1-2.1O-‘4 mol cell-’ h-l) in saline cultures, suggesting the importance of biologi- cal production in the photic zone. The photochemical

Analytical techniques for monitoring hydrogen peroxide

Various analytical techniques have been used for the determination of Hz02 in environmental, biologi- cal and synthetic media. Many of these techniques have used flow injection analysis (FIA) to improve sample throughput and precision. General difficul- ties encountered with the analysis of such a transient

TABLE 2. Techniques and detection limits for the determination of hydrogen peroxide in environmental matrices

Technique

Spectrophotometry

Fluorimetry

CL (peroxyoxalate)

Voltamperometr-y

Matrix LOD (nM)

Seawater 12 Rainwater 140 River water 10

Groundwater 4.6 Seawater 4 Snow 3

Rainwater 15

Rainwater 5

Definition

3 SD. of blank Not given 3 S.D. of blank

3 S.D. of blank 3 SD. of blank Not given

S/N = 3

Not given

Methodology Ref.

Flow-injection 20 Flow-injection 21 Static 22

Static 24 Static 25 Flow-injection 26

Flow-injection 33

Static 47

trends in analytical chemistry, vol. 11, no. 70, 1992 381

and reactive species include the choice of a reliable standardization method, the stability and purity of reagents and low level interferences (e.g. by organo- peroxides). Table 2 shows the range of techniques that have been used for the determination of Hz02 in waters.

Spectrophotometry

The simplest and most widely available technique for monitoring Hz02 is the use of complexing ligands with spectrophotometric detection. Frew et al. [18] presented five oxidation procedures for the determi- nation of organic peroxides and Hz02 but found that only the horseradish peroxidase catalysed oxidation of 4-aminoantipyrene and phenol was suitable for the selective determination of H202, and then only in the pM range. This level of sensitivity was also attained by reducing copper(I1) to copper(I) with Hz02 and complexing with 2,9-dimethyl- 1 ,lO-phenanthroline [ 191. Hydrogen peroxide in rainwater, seawater and lakewater has been determined spectrophotometri- tally by the catalysed oxidation of N-ethyl-N-(sul- phopropyl)aniline to form a coloured condensation product in the presence of 4-aminoantipyrene and peroxidase [20-221. This method has been used for Hz02 analysis at sea with ti detection limits [20]. FIA was utilised to increase sampling rates to 80 h-l and the method was unaffected by manganese(I1) and iron ions which did, however, interfere with a luminol-Hz02 chemiluminescence technique [21].

In clinical applications Hz02 is often determined as the end product of a selective reaction between an oxidase enzyme and its substrate, for example the determination of glucose in urine [23]. The Hz02 produced from the glucose oxidase catalysed oxida- tion of glucose was determined by measuring the colour change of thio-Micheler’s ketone [4,4’-bis(di- methylamino)thiobenzophenone] in the presence of iodide and molybdate.

Fluorimetry

Fluorimetric methods have been used extensively to determine H202. Holm et al. [24] made use of the fluorescent quenching of scopeletin by reaction with Hz02 catalysed by horseradish peroxidase. Ground- water samples were analysed with a limit of detection (LOD) of 20.2 nJ4. These workers used blanks which had been passed through a column of MnO2 (instead of the more traditional method of adding catalase) to

reduce the background levels of Hz02 in dis- tilled/deionised waters (5-2.5 I&!). Storage for >l h causes serious errors and irreproducible results. This fluorescence method has been used by Zika et al. [4] to determine Hz02 in seawater. The dimerisation of (p-hydroxyphenyl)acetic acid by hydrogen peroxide in the presence of peroxidase was used by Miller and Kester [25] to analyse for Hz02 in seawater using only one mixed reagent but marine organic com- pounds were found to interfere. The same method was adapted to analyse snow samples with pressur- ised porous membrane reactors introducing the re- agents in a reproducible manner via a flow system [26]. A similar acid, hydroxyphenylpropionic acid, was used by Roulier et al. [27] to quantitate levels of biologically important cholines in seawater by oxi- dising the choline to betaine and Hz02 with the enzyme choline oxidase. This indirect method for choline was sensitive enough for nM tracing of the compound in coastal seawater. The catalytic effect of manganese tetrakis(sulphophenyl)porphyrin (MnTPPS4) on the fluorescence reaction of ho- movanillic acid and Hz02 was found to be similar to that of horseradish peroxidase, which is relatively expensive and unstable [28]. Kieber and Helz [29] successfully verified a fluorimetric method by the ultimate calibration method of iodimetric titration. This was applied to natural samples of river and estuary water. Genfa et al. [30] investigated the effect of various substrtes on fluorogenic reactions using FIA and UV light to “photodevelop” the product (instead of enzyme mediated oxidation). 4-Hy- droxyphenylacetic acid was found to be the most sensitive substrate but the reaction had to be buffered at high pH while the reaction with 4-hydroxyquino- line-2-carboxylic acid (kynurenic acid) was optimal at pH 6-6.5 with a similar LOD.

Chemiluminescence

The major disadvantage of the spectrophotometric and fluorimetric techniques is that light is scattered in the sample between the excitation source and the light detector, reducing the signal to noise ratio and, consequently, the detection limit is degraded. This problem is much less apparent with chemilumines- cence (CL) methods where the light is emitted from the reaction itself and there is no external light source However, liquid phase CL methods have not been extensively applied to environmental analysis. This is in contrast to the well known gas phase CL reac- tions used for environmental monitoring, for exam-

382

ple the nitric oxide-ozone reaction. The two major reasons for this are the lack of suitable commercial instrumentation and the limited number of selective liquid phase CL reactions available. Peroxyoxalate CL is the most efficient liquid phase CL process with a quantum yield of 0.2 to 0.3 einstein mol-’ compared to luminol and lucigenin with yields of 0.0 1 [3 11. The Hz02 oxidises aryl oxalate esters to excited states which transfer energy to a suitable fluorophore (sen- sitiser) which then emits its characteristic fluores- cence profile. The limitations of this system result from mixing problems with aqueous samples as the peroxyoxalate must be dissolved in organic solvents. Enzymatically generated Hz02 from oxidases has been used to determine glucose by FIA in urine using rhodamine B as the fluorophore [32]. Van Zoonen and co-workers [33,34] developed a flow-injection system with a solid-state peroxyoxalate CL reactor for H202in rainwater [33] and glucose in blood [34], attaining low nM detection limits, wide linear ranges (6 orders of magnitude) and rapid analysis (100 h-l). Katayama et al. [35] used a similar system with a reported LOD of 3 ti using sulphorhodamine 101 as the fluorophore instead of 3-aminofluoranthrene [33] or perylene [34].

Luminol (5amino-2,3-dihydro- 1,4-phthalazi- nedione) CL has been used for many applications, for example the determination of various trace metal catalysts [36];

Luminol + Hz02 + catalyst + base = 3-aminophthalate + light

Horseradish peroxidase was used as the catalyst to determine Hz02 to @4 levels with an FIA manifold [37]. Peroxidase was also used by Nieman [38] by immobilising the haeme-containing species on silica particles to reduce the number of flow channels, but this did not match the detection limit of 3 nJ4 attained with Olsson’s earlier system using similar reagents [39]. Cobalt has been used most successfully to de- termine H202 with home-made or modified instru- mentation [40,41]. Gonzalez-Robledo et al. [40] used a stopped-flow technique for kinetic studies and routine analysis in a synthetic matrix. However the luminol reaction has not been used to date for the determination of Hz02 in natural waters.

Analogues of luminol have been used for clinical applications with varying degrees of success. Isolu- minol was found to give a better result in terms of signal-to-noise ratio but antioxidants were found to interfere by scavenging oxygen radicals which quenched the chemiluminescence and necessitated

trends in analytical chemistry vol. 17, no. 10, 1992

the use of high-performance liquid chromatography for biological samples [42]. Malehorn et al. [43] used lucigenin in the presence of the non-ionic surfactant Brij-35 to increase sensitivity but the level of detec- tion of 330 nJ4 was not as good as that achieved using luminol.

Electrochemical techniques

Various electrochemical techniques have been used to determine H202. A stable grape-tissue based sensor was developed by Wijesuriya et al. [44] but detection limits were only in the 10 PM region. Improved LODs were attained by using modified electrodes, e.g. a glassy carbon electrode coated with copper heptacyanonitrosylferrate(I1) [45] (400 nM), a horseradish peroxidase-modified electrode [46] and a modified tin oxide electrode [47] (both 10 ni%4). Lagrange and Lagrange [48] compared the use of a polarographic method with Hz02 reduction at a mer- cury dropping electrode (200 nM) and a voltam- perometric oxidation on a rotating disk electrode n&Q. Both methods were applied successfully rainwater samples.

(5 to

Future trends for marine monitoring

Hydrogen peroxide is an important parameter to monitor in the marine environment, particularly in the coastal zone, because of its involvement in regu- lating the redox state of seawater [49], in the photo- chemical oxidation of dissolved organic matter and, potentially, its use in wastewater treatment. In order to determine Hz02 in seawater, with natural levels in the nM range, a sensitive method of analysis is re- quired. The procedure must also be selective and the instrumentation suitable for shipboard deployment.

The luminescence techniques described above of- fer the best combination of sensitivity and robust- ness, with the added attraction of speed of analysis for reactive species such as H202, particularly when used in conjunction with FIA. The luminol reaction has already been shown to be suitable for the ship- board monitoring of trace metals [iron and man- ganese] in the marine environment [50,51]. For the determination of Hz02 however, the labile nature of the analyte creates additional challenges in terms of selectivity, the inability to preconcentrate, the pro- duction of suitable standards and the availability of a suitable reference method.

trends in analytical chemistry, vol. 11, no. 70, 1992 383

Acknowledgements K. Toei, T. Tamaru and M. Zenki, Anal. Sci., 6 (1990) 149.

The authors would like to thank the Natural Envi-

ronmental Research Council for financial support (DP) and Kenneth Mopper (Washington State Uni- versity) and Oliver Zafiriou (Woods Hole Oceano- graphic Institute, MA) for their helpful comments.

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384 trends in analytical chemistry, vol. 17, no. 10, 1992

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Paul Worsfold is the Professor of Analytical Science in the Department of Environmental Sciences, Uni- versity of Plymouth, Plymouth, PL4 8AA, UK. His major research interests are environmental analy- sis, particularly field monitoring, process analysis, flow injection techniques and molecular spectros- copy

David Price is a NE/X funded research student in the same department carrying out research on the application of flow injection techniques with chemi- luminescence detection for marine monitoring. Fauri Mantoura is an individual Merit Promotion Senior Principal Scientific Officer at Plymouth Ma- rine Laboratory; Prospect Place, West Hoe, Ply- mouth, UK. His research interests include the development of analytical techniques for the deter- mination of organic and nutrient species in seawater and their biogeochemical cycling.

Lasers in mass spectrometry

John Gormally Salford, UK

Over about the last twenty years, the use of laser techniques has become increasingly common in the field of mass spectrometry. Here, we give a brief overview of the varied applications of lasers in this area. These de- velopments are far from complete and they are expected to continue. However, laser methods have already made valuable contri- butions to problems of mass analysis and it seems likely that they are here to stay.

Introduction

A substantial body of research in the field of mass spectrometry (MS) has been directed towards in- creasing the range of samples to which mass analysis can be applied. The basic MS technique is limited by the requirement that samples have to be pure and reasonably volatile at temperatures low enough to avoid sample decomposition. Over the past ten years, the application of lasers has contributed to the re- moval of some of these limitations.

The two roles of the laser

Laser desorption

Lasers are used in MS for two quite different purposes. Laser desorption refers to the process whereby molecules are ejected from a solid surface

following irradiation with a pulsed laser beam. By this means, it is possible to transfer involatile species from the solid to the gas phase within a mass spec- trometer, allowing ionization and mass analysis to take place. The types of laser used range from pulsed UV lasers, such as the frequency-quadrupled Nd:YAG laser emitting at 266 nm, to pulsed carbon dioxide lasers which emit in the IR at a wavelength of 10.6 pm. In general, the UV laser beams can be focused down to a smaller spot, and they are more likely to lead to the ejection of ions along with desorbed neutral molecules. What is desorbed de- pends very much upon the power density at the target of the desorbing beam. Irradiation of a solid with the tightly focused beam from a pulsed UV laser can cause photoablation and plasma formation. The laser microprobe technique employs lasers in this way, and its varied analytical applications have been reviewed in this journal [l]. Irradiation power densities can exceed lo9 W cmT2 over a spot which may be only a few pm in diameter. This would lead to the destruc- tion of large, fragile organic molecules but permits the elemental microanalysis of surfaces, with a high spatial resolution. At power densities below lo6 W cm-*, laser desorption is often accomplished with little or no molecular decomposition, making it par- ticularly attractive for use with samples which con- tain labile molecules. The most impressive demon- stration of the potential of this technique appeared in 1988 when Karas and Hillenkamp [2] reported their development of matrix-assisted UV laser desorption which enabled them to obtain the mass spectrum of bovine serum albumin (approximate molecular weight 67 000). Subsequently, matrix-assisted laser desorption within a time-of-flight mass spectrometer

0165~9936/92/!$05.00 0 1992 Elsevier Science Publishers B.V. All rights reserved