Analytica Chimica Acta 421 (2000) 35–43

Sensitive reversed-phase liquid chromatographic determination ofhydrogen peroxide and glucose based onternary vanadium(V)-hydrogen peroxide-

2-(5-bromo-2-pyridylazo)-5-diethylaminophenol system

Sławomir Oszwałdowski, Robert Lipka, Maciej Jarosz∗Department of Analytical Chemistry, Faculty of Chemistry, Warsaw University of Technology, ul. Noakowskiego 3, 00-664 Warsaw, Poland

Received 6 April 2000; received in revised form 9 June 2000; accepted 13 June 2000

Abstract

The ternary titanium(IV), vanadium(V) and molybdenum(VI) complexes with H2O2 and 5-Br-PADAP were investigated byspectrophotometry (Vis) and chromatography (LC). Occurrence of two different ternary titanium(IV) complexes with hydrogenperoxide and 5-Br-PADAP (limited by concentration of methanol in the sample) was established. Stability of Ti(IV) and V(V)ternary complexes under spectrophotometric and chromatographic conditions was discussed. Two liquid chromatographicmethods for the determination of hydrogen peroxide based on the Ti(IV) and V(V) ternary complexes were developed.Calibration curves are linear within wide ranges of hydrogen peroxide concentration: 0.02–5.00 and 0.15–5.00mmol l−1 withdetection limits 0.016 and 0.150mmol l−1 for vanadium(V) and titanium(IV) systems, respectively. The method based onvanadium(V) system was used for the determination of H2O2 traces in rainwater, generated in water subjected to sonicationand on the surface of the plant leaf (Plantago lanceolataL.), as well as for indirect determination of glucose in a humanserum. © 2000 Elsevier Science B.V. All rights reserved.

Keywords:Binary and ternary complexes of titanium(IV), vanadium(V) and molybdenum(VI);2-(5-Bromo-2-pyridylazo)-5-diethylaminophenol; RP-LC determination of hydrogen peroxide and glucose; Reversed phase liquidchromatography

1. Introduction

Hydrogen peroxide plays an important role in theatmospheric and biochemical processes. The atmo-spheric hydrogen peroxide is formed mostly by therecombination of hydroperoxyl radical (HO2

•), and inrainwater or cloudwater H2O2 may be produced byboth dark and photochemical reactions. It also plays a

∗ Corresponding author. Tel.:+48-22-660-7408;fax: +48-22-660-7408.E-mail address:mj@ch.pw.edu.pl (M. Jarosz).

key role in photochemical process that led to the ozoneformation and is principal oxidant of S(IV) to S(VI)in most atmospheric aqueous systems (especially atpH less than 5). The last process is an important path-way to the acid rain formation ([1] and referencestherein). For these reasons the atmospheric concentra-tion of hydrogen peroxide has extensively been stud-ied for over 30 years, especially in the USA, Canada,West and South Europe, Japan and Hong Kong [1–5].Unfortunately, there is lack of such a data for Mid-dle Europe. Hydrogen peroxide plays also importantrole in living organisms. It is formed in breathing pro-

0003-2670/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved.PII: S0003-2670(00)01031-X

36 S. Oszwałdowski et al. / Analytica Chimica Acta 421 (2000) 35–43

cess, and as a harmful by-product is quickly deacti-vated by enzymes (e.g. catalase). It takes part in themechanism of cell protection, where it is produced byoxidases-components of antibodies [6] and is formedduring enzymatic reaction of glucose with oxygen inthe presence of glucose oxidase or even without thisenzyme, when process is of photochemical character(λ=254 nm, pH=0.8) [7]. It was also found in watersubjected to sonication [8].

For the determination of hydrogen peroxide usu-ally spectrophotometric [4,5,8–28], titrimetric [8,29],electrochemical [8,29–32], fluorescence or chemilu-minescence [7,33–37] and chromatographic [3,38–41]methods are used.

Spectrophotometric methods for the determinationof hydrogen peroxide are based on: (i) oxidation ofmetal cations [9,10], (ii) reaction with chromogenichydrogen donor (or donors) in the presence of cat-alysts [12–16], (iii) mixed ligand complexes forma-tion. Spectrophotometric methods based on binarymetal-H2O2 systems are rather not sensitive [9].Ternary complexes of metal ions with hydrogenperoxide and chromogenic reagent are much morepromising and a number of ternary peroxo metalchelates have been used for the determination ofhydrogen peroxide [4,5,17–27].

Among titrimetric methods, the one based on thereaction with I− or MnO4

− is very simple and pre-cise, but the results strongly depend on the presenceof oxidants or reducing agents [29].

Electrochemical methods used for the determinationof hydrogen peroxide are not very sensitive. Their de-tection limits usually are about 0.1mmol l−1 [31,32].Much more sensitive are luminescence or fluorescencemethods. In these cases detection limits are often lowerthan 0.01mmol l−1 [33–37].

Chromatography was successfully applied for thedetermination of hydrogen peroxide traces in the pres-ence of organic peroxides [2,38–41], but sensitivitiesof these methods strongly depend on the used detector.

The aim of the work was to find ternary (M–H2O2–R, where R is chromogenic reagent) coloredsystem as a base for sensitive, selective and rapidchromatographic method for the determination of hy-drogen peroxide. Among many organic reagents usedfor RP-LC separation of metal complexes [42–44], themost perspective seem to be pyridylazo ones. Theyform stable ternary complexes with metal ions and

hydrogen peroxide [4,22–24] used for the spectropho-tometric determination of both hydrogen peroxide andmetal ions [43,45–47] especially with Ti(IV), V(V),Zr(IV), Nb(V), Mo(VI), Hf(IV), Ta(V), W(VI) andU(VI). As a chromogenic reagent, heterocyclic azoone -2(5-bromo-2-pyridylazo)-5-diethylaminophenol(5-Br-PADAP) was chosen.

2. Experimental

2.1. Apparatus

The chromatographic system was composed ofthe Hewlett Packard HP 1100 liquid chromatograph(Waldbronn Anal. Div., Germany) consisted withG1310A isocratic pump, G1315A diode array de-tector (DAD), a Rheodyne Model 7725i injectionvalve equipped with a 20ml sample loop (Cotati,CA, USA) and analytical column. Following columnswere used: Nucleosil (125 mm×4.0 mm, 5mm),(Macherey-Nagel, Düren, Germany) containing theLiChrospher 100-5-RP-18 e.c. (Merck) stationaryphase, Nucleosil C18 e.c. (125 mm×4.0 mm, 5mm)and Zorbax SB C18 (250 mm×4.6 mm, 5mm) (DuPont, Rockland Technologies, Inc.). The chromato-graphic system was controlled by Pentium MMX(200 MHz, 64 RAM) PC computer equipped witha LC Chemstation 2D (Hewlett Packard) program,which also stored and handled the data. Before in-jection samples were filtered using the syringe dis-posable filter Chromafil 0.45mm, 15 mm diameter,(Macherey-Nagel, Düren, Germany). The absorptionspectra were recorded on a Specord spectrophotome-ter (Zeiss, Jena) with 10 mm glass cells controlled byPC. For pH measurement, an ELPO-N517 pH meter(Poland) was used. Samples were incubated usinga water bath (VEB MLV 8, Germany). Ultrasoundwas generated in an ultrasonic bath (Sonic-6 bath,Polsonic, Poland; output power 3×80 W, vibrationfrequency 40 kHz). Blood samples were centrifugedusing a laboratory centrifuge (MPW 342, Poland).

2.2. Reagents

All of the reagents used were of analytical-reagentgrade. A stock solution of hydrogen peroxide (POCh,Gliwice, Poland) of concentration 1.5×10−3 mol l−1

S. Oszwałdowski et al. / Analytica Chimica Acta 421 (2000) 35–43 37

was daily prepared and kept in a polyethylene flaskin the dark and cold place. The concentration of hy-drogen peroxide was checked by iodometric titration.2-(5-Bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP) (Fluka, Buchs, Switzerland) was doublyre-crystallized from methanol–water medium. A so-lution of concentration 5×10−3 mol l−1 in methanolwas used. Tetrabutylammonium bromide (TBA+Br−)solution (ca. 5×10−2 mol l−1) was prepared by dis-solving about 8.0 g of TBA+Br− in 250 ml of water.Standard solutions (0.1 mg ml−1) of Ti(IV), V(V),Ni(II), Zn(II), Zr(IV), Nb(V), Mo(VI), Sb(III), Hf(IV),Ta(V), W(VI) and U(VI) were prepared accordingto [9]. The used glucose preparation (p.a.) was sup-plied by Polfa Kraków (Poland). Glucose oxidase(from Aspergillus niger, Sigma, Aldrich), solution(200 U ml−1) was kept in refrigerator. Isoamyl alcohol(p.a., POCH Gliwice, Poland) was used for extrac-tion. Acetonitrile (ACN) of HPLC grade (Lab-Scan,Dublin, Ireland) and water deionized and double dis-tilled were used throughout (water should be freshlydistilled — storing significantly increases blank).

2.3. Eluents for the determination of hydrogenperoxide based on Ti(IV)-H2O2-(5-Br-PADAP) andV(V)-H2O2-(5-Br-PADAP) systems

Ti(IV)-H 2O2-(5-Br-PADAP) complex was elutedusing the methanol/water (80:20, v/v) mobilephase containing 5×10−4 mol l−1 of TBA+Br− and5×10−5 mol l−1 of 5-Br-PADAP. pH of the eluentwas adjusted to 4.0 by means of diluted hydrochloricacid and ammonia solutions.

For the determination of hydrogen peroxide and glu-cose based on the system V(V)-H2O2-(5-Br-PADAP)the mobile phase was acetonitrile–water mixture(50:50, v/v) of pH 1.8 adjusted by means of perchloricacid.

Eluents were degassed by argon bubbling through-out the analysis (at least during 20 min before first in-jection).

2.4. Determination of hydrogen peroxide based onTi(IV)-H2O2-(5-Br-PADAP) system

To a 10 ml calibrated flask was poured 25ml ofTi(IV), an appropriate volume of sample solution

containing hydrogen peroxide in concentration up to5mmol l−1, 50ml of the 5-Br-PADAP solution and5 ml of methanol and 2 ml of acetic buffer (pH=6.0).The flask was filled with water to the mark. After10 min a 20ml portion of the solution was injectedonto the column. The flow rate was 1 ml min−1 andthe eluate was monitored at 580 nm using a DAD de-tector. The concentration of hydrogen peroxide wasdetermined by measuring the peak area.

2.5. Determination of hydrogen peroxide based onV(V)-H2O2-(5-Br-PADAP) system

To a 10 ml calibrated flask 1 ml of hydrochloricacid solution (1 mol l−1), 50ml of V(V), 50 ml of the5-Br-PADAP solution and appropriate volume of sam-ple solution containing hydrogen peroxide in concen-tration up to 5mmol l−1 were transferred. The flaskwas filled with water to the mark. After 30 min a 20mlof the solution was injected onto the column. The flowrate was 1 ml min−1 and the eluate was monitored at590 nm.

2.6. General procedure for glucose determination

To a 10 ml volumetric flask 5 ml of phosphate buffer(pH=5.7), appropriate volume of glucose solution and400ml of the glucose oxidase solution were added.The solution was incubated at 37◦C for 15 min in awater bath. Then 100ml of concentrated hydrochlo-ric acid 100ml of V(V) solution and 100ml of the5-Br-PADAP solution, respectively, were added andthe solution was filled with water to the mark. After30 min a 20ml portion of the prepared sample was in-jected onto the column.

2.7. Samples

Determination of hydrogen peroxide in all analyzedmaterials was performed by means of the methodbased on V(V)-H2O2-(5-Br-PADAP) system.

Samples ofrainwater were collected in polyethy-lene container during event and immediately analyzed.

For the evaluation of the effect of external acousticfield on the formation of hydrogen peroxide, samplesof distilled water (ca. 9 ml) in 10 ml-calibrated glassflasks were placed into the ultrasonic bath (at the

38 S. Oszwałdowski et al. / Analytica Chimica Acta 421 (2000) 35–43

bottom and about 6 cm from the bottom). Sampleswere subjected to sonication for 15 min.

In order to determine hydrogen peroxide generatedby the leaf (Plantago lanceolataL.) surface, 10 ml ofwater containing 1 ml of hydrochloric acid (1 mol l−1),50ml of V(V) and 50ml of the 5-Br-PADAP solu-tions, respectively, was transferred to the beaker. Freshleaf, just picked, was poured into the beaker and af-ter 30 min a 20ml of the solution was sampled andinjected onto the column.

Human serum analysis for glucose content was per-formed as follows: samples of blood were centrifugedat 4000 rpm during 10 min. A portion of the obtainedserum (200ml) was transferred into 10 ml calibratedflask and diluted to the mark with distilled water. 50mlof such obtained solution was then analyzed accord-ing to the general procedure for glucose determination(see above).

3. Results and discussion

3.1. Complexes of Ti(IV), V(V) andMo(VI) with 5-Br-PADAP and hydrogenperoxide–spectrophotometric and chromatographicstudy

All the preliminary experiments were carried outusing following concentrations of the reagents: hydro-gen peroxide 10mmol l−1; metal ions 0.5mmol l−1;5-Br-PADAP 100mmol l−1. Both binary and ternary(with hydrogen peroxide) systems of selected metalions: Ti(IV), Zr(IV), Hf(IV), V(V), Nb(V), Ta(V),Mo(VI), W(VI) and U(VI) were examined spectropho-tometrically and chromatographically (RP LC) in awide range of pH: 2–8 (limited by application rangeof a typical C18 column). Spectrophotometric studyclearly showed, that stable ternary complexes withhydrogen peroxide and 5-Br-PADAP form only tita-nium(IV), vanadium(V) and molybdenum(VI).

Among the studied ternary systems the highestmolar absorptivity exhibited vanadium one (ε(H2O2)=3.3×104 l mol−1 cm−1, λmax=590 nm, pH=1.0–1.5).Molar ratio V(V):5-Br-PADAP in this complexdetermined by Bent–French method, 1:1, agreedwith literature data [47]. Full color developmentwas obtained within 20 min after mixing thereagents. Molybdenum(VI) formed only ternary

(in the presence of hydrogen peroxide) complexwith 5-Br-PADAP (ε(H2O2)=1.2×104 l mol−1 cm−1,λmax=640 nm, pH=1.8–2.5). Evaluation of the molarratio Mo(V):5-Br-PADAP in the complex moleculewas impossible, because it was formed only in thepresence of a large excess of 5-Br-PADAP. The for-mation of the chelate was completed within 30 min.

Binary Ti(IV)-(5-Br-PADAP) complex was formedin acidic medium (when hydrolysis of titanium(IV)ions was negligible [48]), for pH below 2.5. The re-action needed at least 20 min to be completed. Un-der such conditions the system was stable; its absorp-tion maximum was situated at 605 nm. Ternary tita-nium(IV) systems were formed and were stable in awide range of pH: 4.0–8.0 (maximum absorbanceswere measured within pH range 4.0–6.0).

Under optimum conditions two different ternarycomplexes of titanium with H2O2 and 5-Br-PADAPwere formed (Fig. 1), depending on methanol

Fig. 1. Absorption spectra of ternary complexes of tita-nium(IV), vanadium(V) and molybdenum(VI) with hydrogenperoxide (10mmol l−1) and 5-Br-PADAP: a — Ti(IV) sys-tem (20% MeOH); b — Ti(IV) system (60% MeOH), Ti(IV)(5mmol l−1), 5-Br-PADAP (30mmol l−1); c — Mo(VI) system,Mo(VI) (5 mmol l−1), 5-Br-PADAP (50mmol l−1) and d — V(V)system, V(V) (10mmol l−1), 5-Br-PADAP (20mmol l−1).

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Fig. 2. 3D chromatograms of Ti(IV)-H2O2-(5-Br-PADAP) systems. (a) sample containing 20% of methanol; (b) sample containing 60%of methanol.

concentration in solution. For its concentrationsbelow 40% prevailed the species absorbing at540 nm (ε(H2O2)=2.0×104 l mol−1 cm−1), forthose above 50% the form absorbing at 580 nm(ε(H2O2)=2.7×104 l mol−1 cm−1). The species couldbe chromatographically separated. Fig. 2 presents3D chromatograms (absorbance versus time andwavelength) of the samples containing 20 and 60%of methanol, respectively. In the first case twobad separated peaks were observed: the first peak(k′=0.49) was assigned to the complex absorbingat 540 nm and the second (k′=0.69) to the complexwith λmax=580 nm. On the second chromatogram(Fig. 2b) the species absorbing at 580 nm distinctlypredominates.

In both complexes molar ratio Ti(IV):5-Br-PADAPwas evaluated by the Bent–French method as 1:1. Oc-currence of two different species was probably due tothe active role of water molecules in stabilization ofternary system. Such hypothesis was confirmed by theresult of extraction study. Removing of water envi-ronment (extraction of the sample solution absorbingat 540 nm, containing 20% of methanol, by isoamylalcohol) led to the formation of the more hydropho-bic species absorbing at 580 nm (λmax of the obtainedextract). The formed complexes are in equilibriumin solution containing methanol in smaller concentra-tion — signals of both reach maximum absorbancein 5 min, but after 45 min only band at 540 nm is ob-served. Such phenomenon can assume, that reactionwith water molecules is rather slow.

3.2. Chromatographic determination of hydrogenperoxide as Ti(IV)-H2O2-(5-Br-PADAP)

Chromatographic experiments were performedwith use of Nucleosil and Lichrospher RP (bothend-cappped) as well as Zorbax SB stationaryphases. Binary Ti(IV)-(5-Br-PADAP) complex wasnot eluted in any case. It was found, that whensimple water–methanol mixtures were used as elu-ents and Nucleosil or Lichrospher RP as stationaryphase, on the chromatograms there were no signalsof ternary titanium species, possibly because of in-teractions between the complexes and ionized silanolgroups (not end-capped) present on the stationaryphase surface. Similar behavior of metal complexesseparated on even end-capped C8 and C18 phaseshas been previously described [49–51]. Modifica-tion of the mobile phase with TBA+Br− allowed toregister peaks of ternary complexes, but they wereasymmetric (symmetry factors,T >3.5; T=W/2Wa;whereW is the peak width at 10% height from thebaseline andWa is the peak front edge width atthe same height). Among tested phases only Zor-bax SB ensured proper conditions for the separationof Ti(IV)-H 2O2-(5-Br-PADAP) complexes. Its sur-face is sterically shielded with diisobutyl substituents[52,53] and probably because of it access of metalchelates to ionized silanols is much more difficultthan in the case of conventional phases, end-cappedby trichlorosilane. TBA+Br−, acting as dynamicmodifier of the stationary phase thanks to hydropho-

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bic interactions with alkyl chains and ion-exchanginginteractions with silanols [54], caused increasing ofthe peak area. It was found, that optimum concentra-tion of TBA+Br− in the eluent is 250–500mmol l−1.Also the addition of 5-Br-PADAP to the eluent inconcentration 10–100mmol l−1 increased stability ofthe ternary system. As a mobile phase water mixtureswith acetonitrile (ACN) or with methanol (MeOH)were tested, and water––methanol system (containing75–80% of MeOH) of pH from the range 4.0–6.0 waschosen. It was evaluated, that under chromatographicconditions the presence of acetonitrile in the eluentcaused decomposition of ternary titanium complex.Such effect was not observed in spectrophotometricexperiments, and it allowed to assume, that ACNmolecules exhibited weaker affinity (than methanol orwater ones) to silanols, what was previously reported([54] and references therein).

The second examined ternary system (of vana-dium(V)) was found as to be much more stable.Each of the stationary phases tested in prelimi-nary experiments ensured good separation of bi-nary and ternary species formed in the systemV(V)-H2O2-(5-Br-PADAP). Also both examined elu-ents, containing methanol or acetonitrile in concen-tration 70 or 50% (v/v), respectively, were suitablefor obtaining satisfactory separation of the complexes(higher concentration of organic solvents in a mix-

Table 1Characteristics of the chromatographic methods for the determination of hydrogen peroxide based on Ti(IV)-H2O2-(5-Br-PADAP) andV(V)-H2O2-(5-Br-PADAP) systems

System Ti(IV)-H2O2-(5-Br-PADAP) V(V)-H2O2-(5-Br-PADAP)

Column Zorbax SB ODS Nucleosil C18 e.c.Retention factor (k′) 0.58 0.90Linear range of calibration graph (mmol l−1)a 0.15–5.00 0.02–5.00Detection limit (mmol l−1) 0.15 0.02

Calibration graphb

a 63.63 99.31R.S.D.a (%)c 0.58 0.39b 12.5 3.4R.S.D.b (%)c 5.2 15.4Correlation coefficient (r) (n=10) 0.99987 0.99996S.D.d 1.69 1.75Test F for r (F1,8,0.05=5.32) 29800 65660

a Detection limit, DL=3×S.D.bl/a; S.D.bl standard deviation calculated using blanks,a the slope of the corresponding calibration graph.b Peak area (y, mA s, where mA absorbance miliunit) vs. concentration of hydrogen peroxide (c, mmol l−1); y=ac+b.c R.S.D.a and R.S.D.b relative standard deviations ofa and b, respectively.d S.D. the mean standard deviation of regression.

ture with water made separation less effective andlower one caused lengthening of the analysis timedue to increasing retention factors of the separatedspecies). Finally water–acetonitrile mixture was cho-sen because of the low back pressure of ACN. Theoptimum pH of the eluent was quite wide, 1.8–6.0;in further experiments mobile phase of pH about 1.8(adjusted by means of perchloric acid, because of thesmall ability of perchlorate to complex formation)was used, because in less acidic media worse peaksymmetry was observed.

For both examined systems calibration curves wereprepared under optimum conditions. Characteristics ofthe developed chromatographic methods for hydrogenperoxide determination is presented in Table 1.

Comparison of the presented data allows tofind, that the method based on the ternary systemV(V)-H2O2-(5-Br-PADAP) is far more sensitive, thanthe second one. It requires relatively short time forcompleting analysis and offers simpler procedure.The chromatographic separation before spectrophoto-metric detection makes this method highly selective.The diverse effect of common anions, cations and fre-quently used masking ligands on hydrogen peroxide(present in concentration 0.1mmol l−1) determinationwas studied and it was found, that chloride, sulphate,phosphate, carbonate and bicarbonate, as well as thefollowing cations: sodium, potassium and calcium up

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to the concentration 1 mmol l−1, V(IV) up to 10 andTi(IV) up to 5mmol l−1 did not interfere. The pres-ence of Fe(III), Mo(VI) and Cu(II) in concentration5mmol l−1 diminished peak areas of about 20, 40 and40%, respectively. This effect was probably caused bythe formation of complexes of these metal ions with5-Br-PADAP because increasing of the concentrationof 5-Br-PADAP allowed to avoid this error. Becauseof the advantages presented above, the method basedon ternary vanadium system was chosen for analysesof real materials: rainwater, water subjected to soni-cation, medium used for washing leaf surface and ahuman serum.

3.3. Analysis of real materials

Analysis of real materials for hydrogen peroxidecontent is very difficult because of strong oxidizingcharacter of determined species. It is necessary torealize, that obtained results in such analyses mostlyreflect only content of residual H2O2, after its equili-bration with all reducing agents present in the system.Taking this into account and usually having only alittle information dealing to the composition of theanalyzed material, the method of standard addition(MOSA) has to be strongly recommended in per-formed analyses. Although it is more laborious thanthe calibration curve method, it minimizes matrixeffect and allows to normalize proportional errorsin situ. This normalization is possible, because thesame procedural operations are performed on bothunspiked and spiked samples and constant (i.e. re-producible) proportional bias is introduced onto thesamples across the dynamic range [55,56,58].

Hydrogen peroxide content in rainwater was con-trolled during about a year (since September 1998 toJuly 1999). The samples were collected in Warsaw(Warsaw University of Technology area); the obtainedresults are presented in Fig. 3.

Concentration of hydrogen peroxide in rainwaterdetermined by MOSA (Fig. 4) varied between 0.7and 20.7mmol l−1. It was observed, that it was higherin summer rains, than in spring and autumn ones. Itagrees with previously published data [1,2] and re-flects photochemical mechanism of H2O2 formationin atmosphere. Very low concentrations of hydrogenperoxide registered in April and May 1999 are prob-ably due to the very cold and cloudy spring.

Fig. 3. Hydrogen peroxide content in rainwater (samples collectedin 1998 and 1999).

Fig. 4. Determination of hydrogen peroxide in rainwater by MOSA(22 April 1998). a — blank; b — sample (1 ml); c — sample (1 ml)spiked with H2O2 (0.06mmol l−1 in 10 ml final volume). I — peakof V(V)-H2O2-(5-Br-PADAP), II — peak of V(V)-(5-Br-PADAP).

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Table 2Hydrogen peroxide content in water subjected to sonication

Concentration of H2O2 (mmol l−1)

Degassed water Non-degassed water

At the bottom 4.5±2.5 2.0±0.9At the surface 2.5±1.1 0.02±0.01

The effect of external acoustic field (ultrasound)on the formation of hydrogen peroxide in water wasexamined using standard ultrasonic bath. Flasks withtested water (non-degassed or degassed by argon bub-bling during 40 min) were immersed in the bath at thebottom and close to the surface. Results of the per-formed experiments are presented in Table 2. It wasevaluated, that hydrogen peroxide content was higherin samples placed at the bottom of the bath, whenacoustic pressure is the strongest [57] and also in de-gassed water, from which species affecting hydrogenperoxide formation (reactive towards free radicals) areremoved.

Idea concerning searching for hydrogen peroxideon the stressed plant leaf surface was generated by theknown phenomenon — production of H2O2 by oxi-dases (components of antibodies) in peroxysomes [6]as the effect of external emergency (in the case —picking up and pouring into acidic medium). Thanksto high sensitivity of the developed method the pres-ence of hydrogen peroxide on such prepared leaf wasconfirmed and its concentration was determined as7.0±1.0 nmol cm−2.

The optimum conditions for the enzymatic reac-tion of glucose with glucose oxidase were describedelsewhere [23]. For the developed method incubationtime was evaluated as 15 min in 37◦C. Under experi-mental conditions calibration graph for glucose deter-mination was linear in the range 0.05–2.00mmol l−1;detection limit for the method was 0.05mmol l−1.Glucose was determined in human serum using ob-tained calibration graph, as well as MOSA. Statisticalcharacteristics [55,56,58] of both methods is pre-sented in Table 3. The obtained results of the analysesof a human serum sample for glucose content werein a good agreement: 81.9±6.7 mg dl−1 (calibrationgraph) and 80.4±8.4 mg dl−1 (MOSA), respectively.The same sample was also analyzed in professionalmedical laboratory (with use of AlcyonTM 300i Anal-

Table 3Statistical evaluation of the standard calibration graph and thegraph of MOSA for the determination of glucose based onV(V)-H2O2-(5-Br-PADAP) systema

Calibrationgraph

MOSA(human serum)

Linear range of the graph(mmol l−1)

0.05–2.00 0.15–0.75

Detection limit (mmol l−1) 0.05 –

Graph parametersa 109.6 110.0R.S.D.a (%) 1.6 2.4b 12.5 63.4R.S.D.b (%) 11.4 1.8Correlation coefficient(r) (n=7)

0.9994 0.9986

S.D. 2.9 1.7Test F for r (F1,5,0.05=6.61) 3842 1807

a Abbreviations and symbols used in the table are the same asin Table 1; probability level 95%.

yser, Abbot Lab. USA; standard spectrophotometricdetermination) and glucose concentration was deter-mined as 86 mg dl−1.

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