Fluorimetric sensor for the determination of fluoride at the nanograms per millilitre level

Analytrca Chrmrca Acta, 234 (1990) 345-352 Elsevler Science Pubhshers B V., Amsterdam

345 - Pnnted

m The Netherlands

Fluorimetric sensor for the determination of fluoride at the nanograms per millilitre level DANHUA Department

CHEN

‘, M.D

LUQUE

DE CASTRO

and M VALCARCEL

*

of Analytrcal Chemrstry, Umoerslty of Cordoba, 14004~Cordoba (Sparn) (Received

4th December

1989)

ABSTRACT A rapld, sensitive and selective method IS described for the determmatlon of traces of fluonde m real samples based on the integration of retention and fluorescence detection (h,, = 335 nm, h,, 405 nm) of a ternary complex [urconmm(IV)-Calcem Blue-fluoride) using a conventional flow cell packed with an anion-exchange resin A study of a large number of expenmental vanables (flow-mjectlon configuration, type of support, elutmg carrier, sample pH, etc ) allowed the development of an optmuzed, highly selective deternunatlon of fluonde with an analytIca concentration range of l-40 ng ml-’ (r.s.d 1%) with a samphng frequency of 30 h-’ A cntlcal comparison with a probe sensor using the same chermcal system showed the described flow-through sensor to be clearly supenor Keywords

Fluonde

Much effort is being devoted to the development of (bio)chemical sensors in analytical chemistry [l-6]. Nevertheless, there are a surprising number of papers describing such devices with serious drawbacks (lack of sensitivity and selectivity, u-reproducibility, short lifetimes, sluggish response, etc.) which make their practical application useless. For this reason, the development of (bio)chemical sensors that can actually be used in solving real analytical problems is very important. (Bio)chemical sensors can be classified according to several criteria, namely external shape, operational mode and relationship between the sensitive microzone and the transducer. Two broad groups can be distinguished: probe types m which the sensitive microzone and the transducer are connected, and which can operate in a batch mode or be incorporated into a continuous flow system, and flow-through types in which the immobilized ’ Permanent address Department verslty, Wuhan, Chma. 0003-2670/90/$03.50

of Chermstry,

Wuhan

0 1990 - Elsevler Science Pubhshers

Um-

BV

species (rmcrozone) is integrated in the detector and which can only operate in a continuous manner. Both types are being increasingly integrated into flow-injection analysis (FIA) configurations m order to exploit the advantages of both approaches and even to obtain combined benefits. Probe-type (bio)sensors such as enzyme electrodes [7], chemically modified electrodes [8] and optrodes (optical fibres) [9-121 have been used in flow-injection manifolds. The integration of reaction (and/or retention) and spectroscopic detection in contmuous-flow configurations involves the immobilization of any of the components of a (bio)chemical reaction (the analyte, reagent, catalyst or reaction product) in a flow cell packed with an appropriate support (ion-exchange resin, bonded-phase silica beads, etc.). There are also a few systems based on the dynamic immobihzation of the analyte [13]. A flow cell containing a permanently immobilized reagent allows the development of various interesting analytical methods

D CHEN

346

such as the photometnc determination of copper [14], the chemilummescent determmation of peroxides [15,16] and peroxyoxalate [17] and the fluorescence determination of sodium, potassium and calcmm [18]. The analytical reaction can occur in the flow-injection manifold and the reaction product IS retained on the support in the flow cell; such 1s the case with the deterrnmatron of iron [19], chromium [20] and bismuth [21]. The immobilizatron of enzymes in flow cells is a promising trend m this field [22-241. Owing to the complex chemistries involved, there are only a few (bio)chemical sensors for inorganic anions. The determination of fluonde, which is very important consrdering Its direct relationship with public health [25], has proved to be difficult in view of the detection limits reported, most of which vary between 10 and 20 ppb [26-341 or are even poorer [35]. An optical-fibre sensor for the determination of 0.5-8 pg ml-’ fluoride was described recently [36], but it takes several hours to construct and is usable for only 15 measurements; moreover, it has long response and regeneration times. A sensitive, reliable and inexpensive sensor featuring a rapid response for the determinatron of fluonde is therefore desirable. Since the main advantage of the method of integrating retention and detection is a much enhanced sensitivity, the aim of this work was to determme fluoride by measuring the fluorescence of the ternary complex of zirconium, Calcein Blue and fluoride [37] retained on an anion-exchange resin, followed by complete elution with 0.4 M hydrochloric acid. This appears to be the first attempt to determine fluoride with a flow-cell type chemical sensor with simultaneous retention, determination and elution.

EXPERIMENTAL

Apparatus Fluorescence measurements were made on a Kontron SFM 25 spectrofluorimeter equipped with a Knauer X-t recorder, a Gilson Minipuls-2 peristaltic pump, three variable-volume Rheodyne Teflon rotary valves with loop volumes of 500 ~1 and a fluorescence flow cell of i.d. 1.1 mm packed

ETAL

with 40-120-pm DEAE-Sephadex anion-exchange resin (Sigma) along the light path. Reagents Sodmm fluoride A stock standard solution of 100 pg F- ml-’ was prepared by dissolving 221.0 mg of analytical-reagent grade sodium fluonde m 1 1 of distilled water. Working standard solutions of l-40 ng ml-’ were made directly by dilution with distilled water. Calcern Blue (CB). A 1 X lop4 M solution was prepared by dissolving 16.1 mg of Calcein Blue (Sigma) m a few drops of 0.1 M sodium hydroxide solution and diluting to 500 ml with distilled water. The solutron was stable for at least 3 days. Zwcomum chloride. A 0.01 M stock standard solution was prepared by dissolving 0.322 g of ZrOCl, (Merck) in 100 ml of 3 M hydrochlonc acid. A 1 x 10e4 M working standard solutton was prepared for daily use by dilutron with dlstilled water. Bmary complex of zwcomum and Calcem Blue. A 2 X lop6 M solution of the binary complex was prepared by placing 5.0 ml of 1 X lop4 M Calcem Blue and 1 x lop4 M zirconium chloride solutrons in a 250-ml volumetric flask, and adding 1.25 ml of 1 M HCl and after 5 min diluting to the mark with distilled water. Procedure Concentrations of l-40 ng ml-’ of fluoride and 2 X 10e6 M binary complex of zrrconium and Calcein Blue were injected simultaneously from the double valves with a sample loop of 500 ~1. The ternary complex was retained on the support after passing through a 60 cm x 0.5 mm 1.d. reactor. When the maximum fluorescence had been obtained, 0.4 M HCl was inJected from the third valve to elute the complex and to restore the baseline.

RESULTS

AND

DISCUSSION

Fluorescence spectra The fluorescence spectra of the binary complex (Zr-CB) and the ternary complex (Zr-CB-F) at pH 2.6 retained on DEAE-Sephadex (Fig. 1) are

DETERMINATION

OF FLUORIDE

AT

ng/ml

LEVEL

341 200

180

the binary and ternary complexes are retained on the resin mainly through physical adsorption, i.e., there is httle chemical interaction between the retained species and the resm.

160

140

120

Flow-injection configurations Three different configurations were tested (Fig. 2). In configuration A, the Zr-CB complex was previously immobilized and only fluoride was mlected from one valve, and the immobilization was fairly easy and rapid, but unfortunately it was unstable on the resin and was gradually washed away by the carrier. Moreover, the signal pro-

60

A)

60 cm

5x10' M 066

I

_

7

200

300

55C

400

excatot~cm wovelength

em~ssm

wovelength

Fig. 1 Excltatlon and enuss~on spectra of the components of the reactlon 1, Resm (DEAE-Sephadex) m the flow cell, 2, 2 x 10m6 M Zr-CB qected from one valve and retamed on the resm, 3, 2X10e6 M Zr-CB and 40 ng F- ml-’ Injected by the double-valve system and retamed on the resm

similar to those obtained without resin in the flow cell, i.e., the maximum excitation and emission wavelengths in both cases appear at 335 and 405 nm, respectively. The formation of the ternary complex increased the fluorescence while the spectra retained the same shape and position. Although the DEAE-Sephadex itself shows little fluorescence, the blank value is very large if the wavelength difference between the exciting and emitted radiation is less than about 30 nm and the exciting radiation is directly reflected to the detector by the resin surface. However, this has no influence on the determination because it is very distant from the maximum excitation and emission wavelengths. The sunilanty between the spectra obtained with and without resin suggests that

B)

I-40ppb C)

Fig 2. FIA conflguratlons tested for the determmatlon of fluoride (A) The Zr-CB complex 1s lmmoblhzed on the resm and fluonde 1s Injected; (B) use of double-valve system for the reaction before detection, second pump and conectlon to a microprocessor for the elutton; (C) use of double-valve system for the reactlon and a smgle valve for elutlon

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348

duced by injected fluoride was very small compared wrth the fluorescence of immobilized Zr-CB itself. Therefore, double valves were chosen for the stmultaneous Injection of the Zr-CB complex and fluoride, respectively, in order to form the ternary complex in the reactor, followed by simultaneous retention and detection in the flow cell packed with DEAE-Sephadex. After the maximum signal had been reached, the retained product was eluted with 0.4 M HCl. The difference between confrgurations B and C is only in the elutron part; the former requires two pumps to operate alternatively (one for the elutton) and a microprocessor for the exact control of time and the elutron volume. The latter is the better choice because tt needs only a complementary valve to introduce the eluting stream after the maximum signal has been reached.

ETAL

Support

Eight different resins were tested as supports under the same expertmental conditrons. When the signal had reached its maximum, the retained species was eluted with 0.4 M HCl to return to the baseline (Fig. 3). Two cation-exchange resins, SPSephadex and Dowex 50, showed small retention capabilities m comparison with the absence of resin. Better retention, although to varying extents, was obtained with the three neutral resins, XAD-2, XAD-4 and XAD-7. However, there was little difference between the ternary and binary complexes m all of the neutral and cation-exchange resins assayed. Dowex 1, QAE-Sephadex and DEAE-Sephadex showed different retention and different fluorescence between the ternary and binary complexes, but the ratio between the fluorescence of the ternary and binary complexes did not match that

20(IIOmNH

18()-

9

16C, -

6 1LCI-

w " z 8 Ln w IT 0 3 _A u_

120

100

80

60

LO

2 20

TIME

(mln)

Fig 3 Influence of the support used m the flow cell Three mJectlons for blank and analytlcal slgnal each recorded from r&t to left 1. Wlthout support, 2, with Dowex 50, 3, with SP-Sephadex, 4, with XAD-2, 5, with XAD-4; 6, with XAD-7, 7, with Dowex 1, 8, with QAE-Sephadex, 9, with DEAE-Sephadex Concentration of Zr-CB, 2 X 10m6 M Concentration of F-. 0 ng ml-’ for blank and 40 ng ml-’ for analytlcal slgnal

DETERMINATION

OF FLUORIDE

AT ng/ml

349

LEVEL

obtained without resin m the flow cell. This indicates that the ternary complex previously formed is partially decomposed on reaching the three anion-exchange resins, and almost completely decomposed on reachmg the neutral resin, which m turn means that the binding strength between the fluoride and the binary complex is relatively weak. DEAE-Sephadex was eventually selected for further studies and applications because of its large retention capacity and the larger difference between the binary and ternary complexes. Previous work [36] suggested that the Zr-CB binary complex is positively charged, but it was largely retained on anionic and neutral ion exchangers, and scarcely retained on the two cation-exchange resins used. It seems reasonable to conclude that the retention forced between the resin and the retained product was due largely to molecular adsorption rather than to electronic charge, provided that the previous assumption of posittvely charged Zr-CB is correct. Resrn level Different packing heights of the resm were tried (Fig. 4). The signal increased with increasing resin level in the flow cell, but the increment was very small after 2.5 mm. Resin heights above 7.5 mm were avoided as the fluorescence em&on above that level could not reach the detector. There was no variation with resin level in the relative fluorescence ratio between the ternary and binary complexes. Chemical condltlons The experiments showed that the optimum acidity for the reaction to form the the ternary complex in the flow-injection configuration is dilute hydrochloric acid of pH 2.6. Zirconium and Calcem Blue previously prepared at the same concentration also favoured the determmatron. A further study of the Zr-CB concentratron m the range of 5 X lo-‘-8 x lop6 M indicated that 2 x 10e6 M was the best compromise between the absolute and relative signal intensities. Lower ZrCB concentrations narrowed the range of the calibration graph and decreased the signal intensity, whereas higher concentrations increased the blank signal excessively. When fluoride and Zr-CB

160 -

n

ILO-

120-

LO-

20-

0-

, I5

d 10

5 -TIME

0 (mm)

Fig. 4. Resm level Concentration of Zr-CB, 2X 1O-6 M Concentration of F- 0 on right,, 40 ng ml-’ on left 1, 1 2 mm, 2, 25 mm, 3, 5.0 mm, 4, 7.5 mm

were used m stoichiometric amounts to compare the relative signal increments in different concentration ranges, the fluorescence of the ternary complex increased proportionally with increasing concentration, but the increase in the fluorescence of the binary complex slowed with increasing concentration (Fig. 5). For this reason, a larger relative signal for fluoride was obtained with higher Zr-CB concentrations. Elutlon The elution of the retained product was effected with different concentrations of hydrochloric acid (Fig. 6). The elution efficiency increased with increasing hydrochlonc actd concentration and the elution was complete at concentrations above 0.3 M. Hydrochlonc acid can reduce the size of the resin to some extent, but it reverted to

D CHEN

ETAL

160 -

;

”

IIME

(mln)

Fig. 5. Zr-CB concentration for the relative analytIcal signal of same stolchlometry as fluonde. Two rephcate mjectlons, recorded from nght to left. (A) 5 X lo-’ M Zr-CB, 0 and 10 ng F- ml-‘; (B) 1 X 10m6 M Zr-CB, 0 and 20 ng F- ml-‘, (C) 15 x 10m6 M Zr-CB, 0 and 30 ng F- ml-‘, (D) 2 x 10m6 M Zr-CB, 0 and 40 ng F- ml-’

60.

LO -

20 -

7

15

TABLE

5 4---

1

Interference dure)

IO

study of (20 ng F-

ml-’

by the proposed proce-

Ion added

Allowable ratlo of foreign Ion to fluonde (w/w) Proposed method

Manual method 1371

ClNO; B,O: so,’ PO2 AsO; OAcco; Fe’+ cl?+ Ca2+ Bazf Fe’+ Cd’+ Mg2+ Zn2+ Mn2+ co2+ N12+ Pb2 +

4oooa 300 500 600 50 40 500 500 40 10 1000” 1000a 100 500 500 200 100 100 500a 250

500 500 50 5 -=z50 5

’ Maximum ratlo mvestlgated.

500

50 50 50 <5 5 50 50

0 TIME

(man)

Fig. 6 Elutlon with hydrochlonc acid of different concentratlons. Ternary complex formed by 2 X 10m6 M Zr-CB and 40 ng Fml-’ and retamed on the resm. Concentration of hydrochlonc acid: (a) wlthout eluent, (b) 0.2 M; (c) 005 M, (d) 0.1 M; (e) 0 2 M; (f) 0 3 M, (g) 0 4 M, (h) 0 7 M.

its normal size when the carrier was passed through the resin again after elution, causing a basehne change over a relatively long time interval. However, a stable baseline was obtained if the retention and elution process took place continuously, I.e., if the retention immediately followed a previous elutron. Therefore, the small change in the resin size had no influence on the determmatron. When the hydrochloric acid concentratron was < 0.1 M, the ternary complex remaining after elution was partially and gradually washed away by the carrier, which contained dilute hydrochloric acid, causing a gradual decrease of the signal.

DETERMINATION

OF FLUORIDE

AT q/ml

351

LEVEL

Study of interferences The influence of 20 cations and anions on the determination of fluoride was studied. The results are summarized in Table 1. The tolerated level was taken as a signal variation withm k 5% m the measurements. Most of the amons and cations listed did not interfere in the determination. The selectivity was much better than that of the manual method. The selectivity was also compared experimentally with that obtained without the sensor; the results were generally similar, but very large differences were encountered for three ions, the ratio of ion to fluoride being 600, 100 and 40, for SO:-, Fe*+ and Fe3+, respectively, for the present method, whereas the corresponding values without the sensor were 50, 10 and 3, respectively. Sample analysts The calibration graph for l-40 ng ml-’ fluoride with three repetitive injections had a slope and intercept of 1.9292 and 1.2634, respectively, and a regression coefficient of 0.9997 for peakheight measurement. The relative standard deviation (r.s.d.) was 1.0% for seven inlections, while the calibration graph was curved at higher fluoride concentrations and the r.s.d. was 2.57% for seven repetitive inlections. Peak-height measurements were therefore preferred. A sample frequency of 30 h-’ was achieved with the proposed configuration for a sample volume of 0.5 ml and a flow-rate The fluoride in tap water was of 0.68 ml min-‘. determined with a different dilution factor by the standard addition method. The results were 146.6, 147.9, 145.0, 146.9 and 140.0 ng ml-’ with an average of 145.3 ng ml-‘. The result agreed well with that obtained by the method based on the same reaction without sensor (149.6 ng ml-‘) and also with that obtained by an ion-selective electrode method (129-203 ng ml-‘) [38], although the tap water was from a different location in the latter instance. Comparrson with the optical-flbre sensor The features of the proposed flow-cell sensor, compared with the optical-fibre sensor based on the same chemical reaction [36], are summarized m Table 2. The greatest advantage of the flow-cell sensor arises from its direct contact with the sam-

TABLE

2

Comparison t1on

of two types of sensors

Parameter

Probe type

Deternunatlon range Detectlon hrmt Time for Preparation Measurement step Regeneration step Reproduclblhty (r.s d.) Sensor hfetlme

0 5-8 pg ml-’ 05pgml-’ Overmght 30 nun 60 nun 15% 15 measurements

based

on the same reac-

Flow-cell

type

l-40 ng ml-’ 1 ng ml-’ (15

mm 1 mm 1 mm

1% > 100 measurements

ple and eluent solutions to be analysed, the retention and elution (regeneration) thus being very easy and rapid. The optical-fibre sensor, however, takes a very long time for the analyte to pass through the membrane to be retained on the resin and back through the membrane in the regeneration step after the measurement. It depends largely on the molecular diffusion rate m relatively stationary solution. The much higher sensitivity of the present method compared with that using the optical-fibre sensor arises from the resin used, because DEAE-Sephadex is better suited than XAD-4 to the determination of fluoride (see Fig. 3).

The authors express their gratitude to the CICyT for financial support (Grant No. PA860146). D.C. is grateful to the Direction General de Investigation Cientifica y TCcnica for financing his stay in Spain.

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