simultaneous determination of o- and p- nitrophenol by first derivative spectrophotometry

SIMULTANEOUS DETERMINATION OF O- AND P- NITROPHENOL BYFIRST DERIVATIVE SPECTROPHOTOMETRY

M. INES TORAL,� PABLO RICHTER, MONICA CAVIERES andWALTER GONZALEZ

Department of Chemistry, Faculty of Sciences, University of Chile, P.O. Box 653, Santiago, ChileE-mail: [email protected]

(Received 14 April 1997; accepted in revised form 18 August 1997)

Abstract. A simple method by first derivative spectrophotometry is proposed for the simultaneousdetermination ofo-nitrophenol andp-nitrophenol. The analytes were separated from samples byliquid-liquid extraction (pH = 7.4) as tetrabutylammonium ion pairs into the 1,2-dichloroethaneorganic phase and subsequently the extracts were evaluated directly by derivative spectrophotometry.Simultaneous determination of both analytes could be carried out using the zero-crossing and thegraphical methods foro-nitrophenol andp-nitrophenol, respectively. The determination ranges werefound to be between 0.115 to 3.00 ppm and 0.130 to 3.00 ppm foro-nitrophenol andp-nitrophenol,respectively. The relative standard deviations were in all instances less than 2.0%. The proposedmethod was applied to the determination of these compounds in fruit juices.

Key words: derivative spectrophotometry, fruit juices, graphic method, nitrophenol, simultaneousdetermination, zero-crossing

1. Introduction

The presence ofo-nitrophenol andp-nitrophenol in vegetable substances such asfood and fruits is indicative of pesticide residues (Daniel, 1979). The chemicalproperties of the organophosphorus and carbamate pesticides, permit the decom-position and hydrolysis of these compounds into plants. Accordingly, it can beexpected that o-nitrophenol and p-nitrophenol appear in these matrix as hydrolysisproducts of some original pesticides. In order to diagnosticate contamination bypesticides, the content of nitrophenols in the above mentioned samples, can be ofconsiderable significance. In this context, simple, sensitive and selective methodsfor the simultaneous determination of p-nitrophenol and o-nitrophenol are in greatdemand.

The simultaneous determination of these compoundshas been carried out by dif-ferent methods including thin-layer-chromatography (Rathore and Begum, 1993),semi-differential cathodic voltammetry (Liu, 1993), high-speed counter-currentchromatography (Drogue,et al., 1991), Capillary HPLC (Ruban, 1993), flow injec-tion method (Leon-Gonzalezet al., 1992), liquid-chromatography-atmospheric-pressure chemical-ionization mass spectrometry (Doerge and Bajic, 1992).

The spectrophotometry is not an appropriate technique for simultaneous deter-mination of these compounds, because the bands of the analytes are overlaping.� To whom correspondence should be addressed.

Environmental Monitoring and Assessment54: 191–203, 1999.c 1999Kluwer Academic Publishers. Printed in the Netherlands.

192 M. INES TORAL ET AL.

Derivative spectrophotometry, which consists in the differentiation of a normalspectrum, offers a useful means for improving resolution of mixtures, because itenhances the detectability of minor spectral features. Consequently, the selectivityand the reproducibility of the results are improved. In this context, this approachwas adopted here. Derivative spectrophotometry has been used directly for simul-taneous determination of both inorganic compounds (Balcerzak and Swiecicka,1996; Toral and Richter, 1995; Toralet al., 1993) and organic compounds (Morelli,1996; Toralet al., 1996), and also for chromatographic detection (Jimena-Garciaetal., 1996) and continuous flow injection reaction/stopped flow detection (Richteret al., 1996; Baranowska and Kozlowska, 1995)

The purpose of this work was to develop a simple, precise and accurate firstderivative spectrophotometric method for determination ofp-nitrophenol ando-nitrophenol. The analytical features of the proposed method were evaluated bymeans of the determination of both analytes in orange, lemon and pear juices.

2. Experimental

2.1. APPARATUS AND INSTRUMENTS

A Shimadzu UV-160 spectrophotometer with 10 mm cells was used for measure-ments of the absorbance and derivative absorption spectra. An Orion ResearchDigital Ion-Analyzer 701 with glass and saturated calomel electrodes was used forpH determinations.

2.2. REAGENT SOLUTIONS

All chemicals were of analytical-reagent grade and the solutions were prepared withhigh-purity water from a NANOpure ultrapure system (Barnstead).O-Nitrophenol(I)andp-Nitrophenol (II ) both >99% pure, Merck were used for preparing stock stan-dard solutions.

2.2.1. Nitrophenol Solutions (100�g/mL)0.100 g of I and II were dissolved separately in 1000 mL of 0.001 M sodiumhydroxide solution. Solutions of 10�g mL�1 and other ranges of concentrationsof the analytes were prepared by diluting the standard solutions.

2.2.2. Tetrabutylammonium Hydrogensulfate (4.5 g L�1) SolutionThe solution was prepared by dissolving 4.5 g of tetrabutylammonium hydrogen-sulfate in 1000 mL of water.

2.2.3. Sodium Peroxide (2% w/v ) SolutionThis solution was prepared by dissolving 20.0 g of sodium peroxide in 800 mL ofwater. The solution was cooled at room temperature and then diluted with water tothe volume, in a 1000 mL standard flask.

SIMULTANEOUS DETERMINATION OFO- AND P- NITROPHENOL 193

2.2.4. Phosphate Buffer Solution (pH = 7.4)A phosphate buffer solution (pH = 7.4) was prepared by dissolving 68.995 g ofsodium dihydrophosphate in 800 mL of water, the pH was adjusted with sodiumhydroxide solution until pH = 7.4 was reached and diluting to the volume in a 1000mL standard flask.

2.2.5. 1,2-Dichloroethane (DCE) Extrapure (sp. gr. 1.25; Merck)

2.3. RECOMMENDED PROCEDURE

2.3.1. General ProcedureTo an aliquot of the standard solution containing less than 90�g of o-nitrophenolandp-nitrophenol, respectively in 150 mL separating funnel, were added 3 mLof 0.5 M phosphate buffer solution (pH = 7.4), 3 mL of tetrabutylammoniumhydrogensulfate solution and adjusted the total volume to 30 mL. The solutionswere mixed and set aside for 3 min. Then was added 5 mL of DCE and the funnelwas shaken for 3 min. When the phases were separated, the organic layer was runout into a dry flask. The zero-order spectra of the DCE extract were recorded overthe range from 200 to 550 nm against a reagent blank, prepared under the sameexperimental conditions, using 10 mm cells. The first derivative spectra over thesame wavelength range, using�� = 4.0 nm were recorded. All the experimentswere carried out at 20�1�C.

2.3.2. Procedure for Determination of I and II in Fruit JuiceThe pulp of the fruit juice was removed by centrifugation. To one aliquot of 45mL was added an aliquot of standard solution containing less than 135�g ofo-nitrophenol andp-nitrophenol, respectively. Then, the general procedure wascarried out and the organic phase was centrifuged. For citric juices, 2 ml of sodiumperoxide were added and then the general procedure was follow.

3. Results and Discussion

3.1. EFFECT OF THE COUNTER ION

The molar absorptivities of theo-nitrophenol and thep-nitrophenol in aqueoussolution are 4491 and 13849 L mol�1 cm�1, respectively. In order to increase thesensitivity and selectivity of the simultaneous determination of these compoundit is necessary to preconcentrate the analytes. In this context, a preconcentrationby liquid-liquid extraction previous to the spectrophotometric measurement wascarried out. Tetrabutylammonium hidrogensulfate was used as counter ion forthe formation and extraction of these compounds at pH = 7.4. Tetramethylam-monium (TMA), Tetraethylammonium (TEA), Tetrabutylammonium hydrogen-sulfate(TBA) were studied as counter ions and their effect on the formation and


the extraction into 1,2-dichloroethane of ion pairs were compared. It was foundthat in the absence of these counter ions, the extraction did not take place andthat the tetrabutylammonium hydrogensulfate (TBA) was the most effective, inconsequence it was chosen as counter ion.

3.2. CHOICE OF EXTRACTANT

Isoamyl alcohol, dichloromethane and 1,2-dichloroethane were tested as extractant.The effect of the extractant on the formation and the extraction of the Nitrophenol-TBA ion pairs at pH = 7.4 was studied. When isoamyl alcohol was used, the bandsof both compounds change with the time, indicating that probably both nitrophenolsare unstable in this solvent. When dichloromethane was used as extactant, the bandsincreased with the time, because this solvent is too volatile a room temperature.Because the bands are not altered, 1,2-Dichloroethane was found to be the bestextractant and it was selected.

3.3. EFFECT OFpH

The optimum pH for the quantitative formation and extraction into 1,2-Dichloro-ethane of each nitrophenol-TBA ion pair was tested by extracting them with 3 mLof 4.5 g L�1 TBA solution in a pH range 2–10 using different buffer solution,such as: acetate, phosphate and borate. The range of optimum pH, in which thesignal was maximum and constant was between 7.0 and 7.6. In this conditionsthe formation of ion-pairs are preserve, because both nitrophenols are present asanions and TBA as cation. At pH <7.0, formation of ion pair was incomplete owingto protonation of the both nitrophenols and at pH >7.6, formation of ion pair wasalso incomplete because the presence of the TBA as cation is underprivileged. Avalue of pH 7.4 was select as optimum.

3.4. SPECTRAL FEATURES

It was found that theo-Nitrophenol, in presence of tetrabutylammonium solutionat pH = 7.4 forms a ion-pair that is extractable into 1,2-Dichloroethane. The zero-order spectrum of the extract exhibits two bands centered at 275 and 350 nm.(Figure 1/curve A). Similarlyp-nitrophenol can be extracted as the p-nitrophenol-TBA ion pair into the organic phase. Its zero-order spectrum shows two bandscentered at 310 and 420 nm (Figure 1/curve B). The spectra of these extracts showthe characteristic band of the phenoxy group.

Wheno-nitrophenol andp-nitrophenol are simultaneously contained in a sam-ple and tetrabutylammonium are present in the aqueous phase, both analytes arequantitatively extracted, as analyte-TBA ion pairs, into the organic phase. Thisextraction process permits to both analytes to be separated and preconcentrated bya factor of 6.


Figure 1. Absorption spectra of DCE extract ofo-nitrophenol-TBA,p-nitrophenol-TBA pairs mea-sured against reagent blank. (A)o-nitrophenol; 0.666�g mL�1, (B) p-nitrophenol; 0.666�g mL�1.All other conditions as in text.

The zero-order spectra of both analytes show that onlyp-nitrophenol could bedetermined directly between 410 to 500 nm. Howevero-nitrophenol do not showprominent peaks for reliable direct absorbance measurements for determination inmixtures (Figure 1). These spectra are not coincident with those reported by Leon-Gonzalezet al., 1992, in similar conditions but using a different spectrophotometer.These authors report thato-nitrophenol do not absorb at 260 nm, consequently theyshow that the simultaneous determination ofo-nitrophenol andp-nitrophenol ispossible by measurement of absorbance directly at 260 and 410 nm, respectively.According to Figure 1, at 260 nm the bands of both compounds are overlapped(both compound, absorb) and the resolution of this mixtures can be only performedby solving a set of simultaneous equations at two wavelengths. In this context, weadopted the derivative spectrophotometry for resolution of these bands.

The order of the derivative, the analytical wavelengths and the�� value, wereoptimized in order to obtain the maximum resolution and reproducibility. To choosethe optimum derivative order, the first and second derivative spectra of the extractscontaining separately the ion-pairs,o-nitrophenol-TBA andp-nitrophenol-TBAwere respectively recorded. As can be seen in Figures 2 and 3, the first deriva-tive spectra are more resolved than the second derivative ones and also a highestsignal/noise ratio were obtained when the first derivatives are used in the determi-nation of both compounds. As the first derivative offers a more valuable means forsimultaneous determination of both analytes, this approach was adopted. Higherderivative orders were also tested, however they yield irreproducible signals. On


Figure 2. First derivative spectra of DCE extract ofo-nitrophenol-TBA,p-nitrophenol -TBA ion pairsmeasured against reagent blank. (A)o-nitrophenol; 0.666�g mL�1, (B) p-nitrophenol; 0.666�gmL�1.

Figure 3. Second derivative spectra of DCE extract of nitrophenol -TBA ion pairs measured againstreagent blank. (A)o-nitrophenol; 0.666�g mL�1, (B) p-nitrophenol; 0.666�g mL�1.

the other hand, it was observed that the noise increases proportionally with thederivative order. The differentiation is obtained digitally, hence a fast value of480 nm/mim was selected.


Figure 4. (a) First derivative spectra of DCE extract ofp-nitrophenol-TBA. (A) 0.333 ppm; (B) 0.666ppm; (C) 0.999 ppm (D)1.333 ppm; (E) 1.666 ppm,�� = 4.0 nm. All conditions as in text. (b) Firstderivative spectra of DCE extract ofo-nitrophenol-TBA. (A) 0,333 ppm; (B) 0.666 ppm; (C) 0.999ppm (D)1.333 ppm; (E) 1.666 ppm,�� = 4.0 nm. All conditions as in text

The selection of the analytical wavelengths was carried out by recording a seriesof first derivative spectra of the extracts containing separately theo-nitrophenol-TBA and p-nitrophenol-TBA, between a concentration range of 0.333 to 2.00ppm, of each analyte. Figure 4a shows that, the graphical method can be used fordetermination ofp-nitrophenol at wavelength of 439 nm. In this wavelength, the


Figure 5. (a) Calibration graph ofp-nitrophenol in presence of 0.666�g mL�1 o-nitrophenol forfirst derivative spectra at 439 nm, using different�� (A) �� = 4.0 nm; (B)�� = 8.0 nm; (C)�� =12.0 nm; (D)�� = 16.0 nm. All conditions as in text. (b) Effect of thep-nitrophenol concentrationover the signal of 0.666�g mL�1 o-nitrophenol for first derivative spectra at 265 nm, using different��. (1)�� = 4.0 nm; (2)�� = 8.0 nm; (3)�� = 12.0 nm; (4)�� = 16.0 nm. All conditions as intext.

distance, hB (Figure 2/curve B) is only proportional to the concentration of thiscompound. Similarly, the measurement of the derivative spectrum at an abscissavalue of 265 nm (hA) (Figure 2/curve A), corresponding to the zero-crossing pointof the derivative spectrum ofp-nitrophenol can be used satisfactory to determine


Figure 6. (a) Calibration graph ofo-nitrophenol in presence of 0.666�g mL�1 p-nitrophenol forfirst derivative spectra at 265 nm, using different�� (A) �� = 4.0 nm; (B)�� = 8.0 nm; (C)�� =12.0 nm; (D)�� = 16.0 nm. All conditions as in text. (b) Effect of theo-nitrophenol concentrationover the signal of 0.666�g mL�1 p-nitrophenol for first derivative spectra at 439 nm, using different��. (1)�� = 4.0 nm; (2)�� = 8.0 nm; (3)�� = 12.0 nm; (4)�� = 16.0 nm. All conditions as intext.

o-nitrophenol (Figure 4b). Further, the zero-crossing point are not dependent of thep-nitrophenol concentration.

In order to select the�� value for derivation, a series of first derivative spec-tra of mixtures of 0.666 ppmo-nitrophenol with increasing concentrations ofp-nitrophenol ranging from 0.333 to 2.00 ppm (Figure 5a and b) were evaluated


under the selected conditions, at different�� values. Similarly, the first derivativespectra of mixtures of 0.666 ppmp-nitrophenol and increasing concentration ofo-nitrophenol from 0.333 to 2.00 ppm were obtained (Figures 6a, 6b). The Figures5a and 6a shows that a good calibration lines are obtained for both compound overthe range of�� from 4 to 16 nm. However, besides getting a good calibration line,it is necessary that the signals for each compound are not be affected by the otherwhich is only possible when a�� value of 4.0 nm is used for differentiation (Fig-ures 6a, 6b). For these reason a�� value of 4.0 nm were selected as optimum. Inthis condition a good reproducibility and signal to noise ratio in the determinationof these species simultaneously is obtained.

3.5. ANALYTICAL FEATURES

The calibration graphs (n = 7) were obtained by plotting the first-derivative valuefor o-nitrophenol hA (265 nm) and hB for p-nitrophenol (439 nm), with�� =4.0 nm, versus the analyte concentrations. The linear regression equations and thecorrelation coefficients, calculated for mixtures ofo-nitrophenol andp-nitrophenolwere:

o-nitrophenolhA = 0.031 C (ppm) – 5 x 10�4 (r = 0.999)

p-nitrophenolhB = 0.040 C (ppm) + 3 x 10�3 (r = 0.999)

where,h is in derivative units and C(ppm) corresponds to analyte concentration inppm.

The determination ranges were between 0.115 to 3.00 ppm and 0.130 to 3.00ppm foro-nitrophenol andp-nitrophenol, respectively.

The detection limits (calculated by using the 3� criterion) were found to be0.034 ppm and 0.038 ppm foro-nitrophenol andp-nitrophenol, respectively.

The relative standard deviations for 10 standard samples containing 0.666 ppmof each compound were 1.8% and 1.7% foro-nitrophenol andp-nitrophenol, respec-tively. The results indicate the valuable analytical features achieved.

In order to establish the ratios at which one analyte can be accurately measuredin presence of the other, the recoveries of samples containing standard solutionsof o-nitrophenol and p-nitrophenol in different concentration ratios were carriedout. The results (Table II) show that the content of each compound can be reliabledetermined, if the concentration ratio is between 1/8 to 10/1 foro-nitrophenol/p-nitrophenol.

Fruit juices were chosen for application of the proposed method. In citric juices,the signal of p-nitrophenol is altered by the presence of ascorbic acid, due to thefact that the nitro group of p-nitrophenol is reduced. This effect was eliminatedby the addition of sodium peroxide previous to the formation of the ion pair. Thiseffect was not observed foro-nitrophenol.


Table IDetermination ofo-nitrophenol andp-nitrophenol in different standard mixtures

Molar ratio Stated concentration / ppm Found concentration�

I:II /ppm � SD (Recovery,%)I II I II

1: 10 0.333 3.33 0.333� 0.003 2.41� 0.023(100.0) (72.4)

1: 8 0.333 2.66 0.331� 0.002 2.63�0.022(99.6) (98.9)

1: 5 0.333 1.66 0.333� 0.002 1.71� 0.021(100.0) (103.2)

1: 2 1.00 2.00 0.984� 0.008 2.01� 0.022(98.4) (100.3)

1: 1 1.00 1.00 0.974� 0.010 1.0� 0.012(97.4) (100.0)

2 : 1 2.00 1.00 2.02� 0.011 0.98�0.009(101.2) (98.0)

3 : 1 1.00 0.333 0.974� 0.010 0.33� 0.003(97.4) (100.0)

5 : 1 3.00 0.666 2.97� 0.031 0.66� 0.005(98.9) (100.0)

8 : 1 2.66 0.333 2.72� 0.030 0.32� 0.030(102.3) (98.0)

10 :1 3.33 0.333 3.24� 0.032 0.33� 0.004(97.4) 100.0

� Mean of ten determination.

Table IIRecovery studies in orange lemon and pear juices, enrichment with standardsolutions ofo-nitrophenol andp-nitrophenol

Fruit Added / ppm Recovery / %�

Juice o-nitrophenol p-nitrophenol o-nitrophenol p-nitrophenol

0.66 1.00 98.2 99.0Orange 1.33 1.66 100.0 100.0

1.66 1.33 100.0 103.0

1.00 0.66 102.0 100.0Lemon 1.33 2.00 98.0 102.8

1.66 1.00 101.2 99.0

0.66 0.66 100.0 100.0Pear 1.33 1.00 98.0 100.0

1.66 2.00 98.2 101.2

� Mean of ten determination.


3.6. RECOVERY IN FRUIT JUICE SAMPLES ENRICHED WITHo-NITROPHENOL ANDp-NITROPHENOL

The proposed method was applied to determination of both compounds in fruitjuice samples enriched with standard solutions ofo-nitrophenol andp-nitrophenolin different ratios. For orange, lemon and pear juices, in all cases, it was necessaryto separate the large amount of pulp by using centrifugation. When this step wasnot carried out, the organic phase shown a high cloudiness and the spectra werestrongly alterated. Derivative spectrophotometry that is particularly advantageouswhen the absorbance changes due to turbidity, in this case the first derivatives donot improved. Results for orange, lemon and pear juices are shown in Table II. Ascan be see in Table II, in all cases a good recovery was obtained.

4. Conclusions

The results demonstrate that a simple, rapid, inexpensive, precise and accuracymethod has been developed for the simultaneous determination ofo-nitrophenolandp-nitrophenol by first order derivative spectrophotometry. The proposed methodwas validated for the quantitative determination of these compounds in fruit juices,such as orange, lemon and pear juices. The results show that a good recoveryand reproducibility were obtained. When the proposed method is applied in juiceenrichment, the recoveries obtained are better than the reported by Leon-Gonzalezet al., 1992. On the other hand, it was demonstrated that, for the simultaneousdetermination of both nitrophenols in citric juices, it is necessary the addition ofsodium peroxide previous to the formation of the ion pair, otherwise reduction of thenitro group of the p-nitrophenol by ascorbic acid is produced and the simultaneousdetermination is not possible.

Acknowledgments

The authors are grateful to the National Fund for Development of Sciences andTechnology (FONDECYT), project 1961024 for financial support.

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simultaneous determination of o- and p- nitrophenol by first derivative spectrophotometry

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