Microchemical Journal 73(2002) 279–285

0026-265X/02/$ - see front matter� 2002 Elsevier Science B.V. All rights reserved.PII: S0026-265XŽ02.00095-4

A continuous flow system combined with a sensing fluorimetrictransductor for the determination ofb-naphthol

S. Ortega Algar, N. Ramos Martos, A. Molina Dıaz*´

Department of Physical and Analytical Chemistry, Faculty of Experimental Sciences, University of Jaen, E-23071 Jaen, Spain´

Received 28 January 2002; received in revised form 19 May 2002; accepted 29 May 2002

Abstract

A single automatic method for continuous flow determination ofb-naphthol based on the enhancement of itsnative fluorescence once the analyte was transitorily retained on-line on a solid support(QAE A-25 resin) is reported.So, a flow-through optosensor was developed using a flow-injection analysis system with solid phase fluorimetrictransduction. KCl(0.15 mol l ) at pH 12.0 was used as carrier solution. To obtain the optimum fluorescence signaly1

the wavelengths chosen were 245 nm(excitation) and 420 nm(emission). The response of the sensor was directlyproportional to the sample volume injected in the studied range 40–1500ml. Approximately one higher order ofmagnitude is achieved in sensitivity when 1500ml are used with respect to the use of 40ml of sample. The sensorwas calibrated for three different injection volumes: 40, 600 and 1500ml, responding linearly in the measuring rangeof 2–60, 0.5–15 and 0.2–5mg l with detection limits of 0.5, 0.09 and 0.05mg l , respectively. The relativey1 y1

standard deviation for ten independent determination is 0.6%(40 ml), 0.9% (600 ml) and 2.3%(1500 ml). Arecovery study was performed onto three different spiked water samples at concentration levels from 1 to 2.5mg l and the recovery percentage from the experimental data ranged between 101"2 and 105"5.y1

� 2002 Elsevier Science B.V. All rights reserved.

Keywords: b-Naphthol; Flow-injection analysis; Spectrofluorimetry; Environmental analysis; Flow-through sensor

1. Introduction

b-Naphthol is a naphthalene derivative com-pound with substituent group at position 2, whichis usually more toxic than those derivatives atposition 1. It is capable of producing severesystemic intoxications. This is a compound ofcommon use in the dyestuffs industry, in pharmacyand as cosmetic. The determination ofb-naphthol

*Corresponding author. Tel.:q34-953-012147; fax:q34-953-012141.

E-mail address: amolina@ujaen.es(A.M. Dıaz).´

is of great importance in quality control and inenvironmental chemistry.Several procedures have been proposed for the

determination ofb-naphthol, involving differentanalytical techniques, such as high performanceliquid chromatographyw1–5x, gas chromatographyw6x, spectrophotometry w7–10x; luminescencemethods including fluorimetricw11–13x, phos-phorimetric w14x and chemiluminescencew15,16xmethods have also been applied to determinationof b-naphthol and in some case, to a bromo-derivativew17,18x.

280 S.O. Algar et al. / Microchemical Journal 73 (2002) 279–285

Recently, the on-line transitory retention of theanalyte(s) on an appropriate solid support placedinside a flow cell has been used for monitoring anintrinsic analytical property from it(them). Thisarrangement has been proved to be a simple,inexpensive and straightforward procedure forimproving important analytical features such assensitivity and selectivityw19,20x.In this arrangement, spectrophotometry has been

the most frequently used detection technique. Itsapplication to the determination of active princi-ples in pharmaceutical analysis has been the mostusual one w21,22x. Only a few methods withluminescence detection have been developedw23–26x, and almost all of them have been applied topharmaceutical analysis.The development of sensitive, fast and inexpen-

sive flow-through sensors in the environmentaland other analysis fields is a challenger alreadystated in an International Conference on AnalyticalChemistry( SAC992) w27x which is worth of beingpaid attention.In this paper a flow-through sensor with fluori-

metric transduction for determination ofb-naph-thol at submg l level is presented. The sensory1

is based on the use of an anion exchange gelpacking in the flow cell, the transitory retention ofthe analyte from the sample plug and the directmonitoring of its native fluorescence. The proce-dure is extraordinarily simple as well as sensitiveand can be applied to monitoringb-naphthol inwaters. This appears to be the first attempt todetermine a phenol derivative compound(b-naph-thol) with a flow-through fluorimetric sensor.

2. Experimental

2.1. Apparatus

A Varian Cary—Eclipse Fluorescence Spectro-fluorimeter (Varian Iberica, Madrid, Spain) wasused to obtain the relative fluorescence intensitymeasurements. The spectrofluorimeter wasequipped with a xenon discharge lamp(75 kW),Czerny-Turner monochromators, two detectors(sample and reference), a R-928 photomultipliertube which is red sensitive(even 900 nm) withmanual or automatic voltage controlled using the

Cary Eclipse software for Windows 95y98yNTsystem.The photomultiplier detector voltage was 700 V

and the scan rate of the monochromators wasmaintained at 600 nm min (a Savitzky-Golayy1

w28x type smoothing with a filter size of 17 wasused). The instrument excitation and emission slitswere set at 5 and 20 nm, respectively. An emissionfilter (295–1100 nm) was used in order to mini-mize the resin background signal at the excitationwavelength (245 nm). The peak heights weremeasured at 420 nm.To generate the flow stream, a four-channel

Gilson Minipuls-3 peristaltic pump with rate selec-tor was used. It was also utilized a RheodyneModel 5041 injection valve with variable sampleloops and PTFE tubing of 0.8 mm i.d.A Hellma 176.052-QS quartz flow-through cell

was used(light-path length of 1.5 mm), whichwas filled with Sephadex QAE A-25 resin. Theresin was placed in the complete light-path of thecell as an aqueous slurry with the aid of a syringe.

2.2. Reagents

All reagents were analytical-reagent grade anddeionized water was used to prepare all solutions.Working solutions of b-naphthol were daily

prepared by suitable dilution of the stock solutionwith deionized water. The concentration of thisstock standard solution of the analyte was6.94=10 mol l and it was prepared by dis-y4 y1

solving the required amount ofb-naphthol(Pan-reac, Barcelona, Spain) in deionized water. Thissolution was stable for 4 days at approximately 58C.KCl (0.15 mol l ) solution at pH value of 12.0y1

was used as carrier solution.The solid support used in this sensor was

Sephadex QAE A-25(Aldrich, Madrid, Spain), inthe H form, as supplied without any pre-q

treatment.

2.3. Procedure

Using a single channel manifold, the samplewas transported, inserted into the carrier stream(KCl 0.15 mol l at pH 12.0) at a flow rate ofy1

281S.O. Algar et al. / Microchemical Journal 73 (2002) 279–285

Fig. 1. Fluorescence spectra(excitation and emission spectra)of b-naphthol 50mg l : (1, 19) on Sephadex QAE A-25 resiny1

(sample volume: 40ml); (2, 29) in aqueous solution. Slit-exi-tation: 5 nm; slit-emission: 20 nm.

Fig. 2. Effect of the carrier solution pH;b-naphthol 50mg l (sample volume: 40ml); flow rate: 1.14 ml min .y1 y1

1.14 ml min , through the flow cell. There, they1

analyte was sorbed on the solid support(SephadexQAE A-25 resin) as its anionic form. The relativefluorescence emission intensity at 420 nm using245 nm as excitation wavelength was continuouslymeasured using slit-width values of 5 and 20 nmfor excitation and emission, respectively.After developing the signal, the desorption of

b-naphthol from the solid support in the flow cellwas performed by the carrier itself, so achievingthe regeneration of the active ion-exchange gelmicrozone and allowing the signal value to returnto the baseline, so remaining it ready for a newdetermination. The peak height was used as ana-lytical signal. Three calibration lines could be usedfor injecting three different sample loops: 2–60,0.5–15 and 0.2–5mg l of b-naphthol for 40,y1

600 and 1500ml, respectively.

3. Results and discussion

3.1. Spectral characteristics

Fig. 1 shows the spectra ofb-naphthol in bothhomogeneous aqueous solution and sorbed onSephadex QAE A-25. A strongly significantincrease in the signal can be observed from thesorption of the analyte on the sensing zone as a

result of its concentration just in the detectionzone.On the other hand, a slight bathochromic shift

(5 nm) in the excitation peak was observed as itssurrounding environment affects the analyte in adifferent way on the resin with respect to theaqueous solution. The total luminescence spectrumshowed that the maximum signal was achieved at245 nm (excitation) and 420 nm(emission). Sothey were chosen as working wavelengths. In theseconditions the signal background was approxi-mately 200.

3.2. Influence of the pH of the carrier solutionand of the sample

This study was carried out using a samplevolume of 40ml. In this system, the effect of pHon the fluorescence intensity has been studied forboth the carrier solution and the sample.The influence of the carrier solution pH was

studied using a 0.2 M NaCl solution and adjustingthe pH values with HCl or NaOH solutions from1.9 to 13.0. This concentration of electrolyte couldinsure the complete elution of the analyte from theresin after developing the peak signal. The sorptionof b-naphthol only occurred at basic pH values,as expected, as its pK value is 9.51w29x. So thepeak signal increases up to reach its maximum atpH value of 12.0(Fig. 2) at which, the analyte is

282 S.O. Algar et al. / Microchemical Journal 73 (2002) 279–285

completely in its anionic form and it is stronglyretained. Beyond this value, the fluorescence signaldecreases probably due to the competition of theOH group by the active sites of the solid support.y

So pH 12.0 was selected as the carrier solutionpH value.In order to optimise the self-eluting action of

the carrier solution used, several electrolytes solu-tions at 0.2 M concentration were tested: NaCl,KCl, NaNO and Na CO . KCl was selected3 2 3

because it gives a higher signal and a lower elutiontime. Next, the influence of its concentration wasstudied from 0.02 to 0.2 M. The increase ofconcentration decreased the signal(due to com-petition produced by the Cl ions) as well they

elution time (47 and 52%, respectively). As acompromise between a high sampling frequencyand a high signal, 0.15 M was the concentrationchosen.On the other hand, the influence of the sample

pH was studied by varying in the range 2.0–13.0.The signal is almost constant from 2.0 to 9.0 anda slight increase(f8%) is achieved between 9.0and 12.0. Higher values also produced a decreasein the signal, similarly at the effect of the carriersolution pH. Therefore, pH 12.0 was selected asthe sample pH value.

3.3. Influence of flow rate

In the study of this variable the 0.29–2.02ml min value range was tested, using an analytey1

concentration of 1mg ml . As the flow ratey1

decreased, both the signal and the elution timeincreased(hence, wider peaks were obtained). Avalue of 1.14 ml min was chosen as a compro-y1

mise between sensitivity and a good samplingthroughput: the signal decreased from 830(flowrate of 0.29 ml min ) to 560 (1.14 ml miny1 y1

flow rate), while the sampling frequency increasedfrom 13 h to approximately 40 h . Thesey1 y1

results are usual in flow-through spectroscopicsensing devices as a consequence of the chromat-ographic process which takes place in the solidsensing zone itself as the ion exchange retentioncompetes against the transport of the analyte bythe carrier stream.

3.4. Study of the influence of the sample volume

The higher the sample volume injected, thehigher the amount of the analyte sorbed on theresin in the detection area itself and, hence, thehigher the sensitivity showed by the solid phaseflow-through sensing system. However, when thesensitivity is not a limiting factor, lower samplevolumes should be used in order to get highersampling throughput.This study was performed using the sameb-

naphthol concentration(5 mg l ), and varyingy1

the sample loop of the injection valve from 40 to1500 ml. A linear increase of the fluorescencesignal RFIs29.6q0.329V (rs0.998, V in ml)was found in the sample volume range tested. Thisseams to indicate a high distribution ratio ofb-naphthol between the solid phase and the solutionsurrounding it.This offers us an interesting feature of the sensor

as it can be taken the advantage of working withdifferent analyte concentration samples just byselecting the appropriate sample loop for injectingand calibrating with the respective sample loop.So, as an example the sensing flow-through devicewas calibrated for the three different sample vol-umes: 40 600 and 1500ml, so obtaining highincreases in sensitivity and decreasing strongly thedetection limits(see figures of merit below).

3.5. Figures of merit

Table 1 contains the figures of merit of themethod proposed for the three calibration volumes,and Fig. 3 shows the three calibration lines. It canbe observed the strong increase in sensitivity pro-vided by the increase in the sample volume inject-ed which allows to work directly at submg ly1

levels using only 1.5 ml of sample and withoutusing any previous preconcentration process of thesample.To evaluate the reproducibility of the method,

ten independent analyses of solutions containing60, 10 and 3mg l of analyte were used fory1

40 600 and 1500ml, respectively. Fig. 4 showsan example of reproducibility(40 ml).The detection limits were calculated by using

the 3s criterion w30x, and values of 0.5, 0.09 and

283S.O. Algar et al. / Microchemical Journal 73 (2002) 279–285

Table 1Figures of merit

Parameter Volume of sample injection(ml)

40 600 1500

Linear dynamic range(mg l )y1 1.7–60 0.31–15 0.17–5

Calibration graphIntercept 9.2 9.7 21.04Slope(l mg )y1 11.5 45.7 102.6Correlation coefficient 0.999 0.999 0.998Detection limit(mg l )y1 0.5 0.09 0.05Quantification limit(mg l )y1 1.7 0.31 0.17RSD (%) (ns10) 0.6 (60 mg l )y1 a 0.9 (10 mg l )y1 a 2.3 (3 mg l )y1 a

Sampling frequency(h )y1 20 20 20

b-Naphthol concentration used to evaluate R.S.D.a

Fig. 3. Calibrations lines obtained from different sample vol-umes injected.(a) 40 ml; (b) 600ml and (c) 1500ml. Inset:Calibration diagram(40 ml).

Fig. 4. Reproducibility for 40ml sample volume(50 mg l );y1

flow rate: 1.14 ml min .y1

0.05 mg l were obtained for the three differenty1

calibrations. The quantification limits were esti-mated by using 10s criterion w31x. It should beemphasized that the method shows a very highersensitivity than other molecular spectroscopic pro-cedures based on spectrophotometricw8x or phos-phorimetric w32x measures. The samplingfrequency obviously is higher for smaller samplevolumes. Using a sample volume of 40ml, a

sampling frequency of 20 h is obtained. Thisy1

value is very suitable in this kind of methodology.

3.6. Effect of foreign species

In Table 2 the effect of various potential inter-fering species, commonly found together withb-naphthol, on the determination of 1mg l (1500y1

ml of sample) is listed. This effect was investigatedby adding a known amount of the tested speciesto the analyte solution. Tolerance level is definedas the foreign species concentration that producedan error not exceeding"5% in the determinationof the analyte.

284 S.O. Algar et al. / Microchemical Journal 73 (2002) 279–285

Table 2Interference study(determination of 1mg l of b-naphthol)y1

Foreign species Tolerated interferentyAnalyte (wyw) ratio

Kq 34 000Cly 31 000Naq 20 000Mg , SO2q 2y

4 1400NOy

3 1200Ca2q 950PO3y4 60NHq

4 40o-cresol, 2-Tert-butyl-4-methylphenol 2500Phenol 950p-Cresol 7502,4-Dichlorophenol 500o-Chlorophenol,p-Chlorophenol, 3,4-Dimethylphenol,p-Nitrophenol 2402,4-Dimethylphenol 804-Methyl-2-nitrophenol,o-Nitrophenol 252,4-Dinitrophenol 10

Table 3Method validation

Water sample Added Mean recovery % Recovery Standard additionconcentration oncentration mean"a calibration graph(mg l )y1 (mg l )y1 tsm slopes(l mg )y1

Jaen city´ 1 1.00"0.04 102"4 81"21.5 1.50"0.04 101"2

Granada city 2 2.0"0.1 105"5 114.5"0.32.5 2.5"0.1 104"4

Torres city 1 1.00"0.05 104"5 145"41.5 1.50"0.05 102"3

Mean of three determinations.a

It can be seen that the procedure proposed showsa high level of tolerance to other species frequentlyfound along withb-naphthol, aso-cresol, 2-tert-butyl-4-methylphenol, phenol,p-cresol and 2,4-dichlorophenol. We should point out this level ishigher than the tolerance to these species found inother methods published for determination ofb-naphtholw9x. It was also studied the effect of someinorganic ions, we found the smaller tolerance forNH , with a ratio of 42(wyw, interferentyanaly-q

4

te). All cationic interferences can be easily sup-pressed by using a cation exchange minicolumnon-line before the sample fills the loop; the analytecannot be retained on the column, while all cations

will be separated from it, and they will notinterfere.

3.7. Method validation

This sensor was applied to the determination ofb-naphthol in water samples. Tap water from Jaen´and Granada city and raw water from the TorresRiver (Jaen province) were selected as represen-´tative samples. They all were found to be freefrom b-naphthol, so a recovery study was per-formed at two concentration levels in each case(Table 3).

285S.O. Algar et al. / Microchemical Journal 73 (2002) 279–285

The standard addition calibration graph methodwas used after checking the presence of a matrixeffect. This effect could be evaluated from theratio of the standard addition calibration graphslope, given in Table 3, and the standard calibrationgraph slope(Table 1). The ratios were 0.79"0.01,1.11"0.01 and 1.41"0.02 for the three samples,respectively.

4. Conclusions

The flow-through system with fluorimetric sens-ing transduction here proposed for the determina-tion of b-naphthol shows very good analyticalfeatures:

a. Sensitivity and selectivity, both as a conse-quence of the selective sorption and(simulta-neously) preconcentration of the analyte in theirradiated detection zone itself( just on the solidsupport).

b. Detection limit lower than those obtained byother luminescence proceduresw15,16,31x in afactor of 10–1000.

c. Reproducibility and rapidity, as usual in theseFIA systems where the carrier solution itselfalso elutes the analyte from the sensing zoneremaining it ready for reutilization.

d. It is extraordinarily simple and inexpensive.None reagent is necessary other than the carriersolution(KClyNaOH).

e. It can be used in routine water analysis.

Acknowledgments

The authors are grateful to Direccion General´de Ensenanza Superior e Investigacion Cientıfica˜ ´ ´of the Ministerio de Educacion, Cultura y Deportes´(Project no. PB98-0301) for financial support.

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