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Continuous-flow chemiluminometric determination of ammonium ion in fertilizers
Tahta,
0039-9140/93 $6.00 + 0.00
Vol. 40, No. 8, pp. 1245-1254, 1993
Printedin Great Britain. All rights remwd
copyright0 1993 Pcrgamon Fvcss Ltd
CONTINUOUS-FL.OW
CHEMILUMINOMETRIC
DETERMINATION OF AMMONIUM IN FERTILIZERS
ION
STERGIOSA. HALVATZISand MJZROPI M. TIMOTHEOU-POTAMIA* Laboratory of Analytical Chemistry, University of Athens, Panepistimiopolis, 157 71 Athens, Greece (Received 3 Augusf 1992. Revised 8 January 1993. Accepted 25 January 1993) Summary-A simple continuous-flow chemiluminometric method for the determination of 0.0540-5.4O~&ml of ammonium ion is described. The method is based on the chemiltinescence generated during the oxidation of ammonium ion by N-bromosuccinimide in alkaline medium. The emission intensity is greatly enhanced if dichlorofluorescein is also present in the reaction solution. The analysis is automated, requires no sample pre-treatment and solutions can be analysed at a rate of 106 solution@ with a relative error of about 2.5%. The method was applied satisfactorily to the determination of ammonium ion in solid and liquid fertilizers.
A wide variety of methods and techniques are available for the determination of ammonium ion. Spectrofluorimetry,‘J molecular emission cavity analysis,3 gas phase molecular absorption spectrometry,4 capillary isotachophoreGs,s ionselective electrodes,‘j conductometry,‘T8 and thermometric titrimetryg are only some examples of the techniques available for the determination of ammonia and ammonium ions. Flow injection systems with gas diffusion or ion exchange modules and spectrophotometric detection are described in the literature.‘“‘* The spectrophotometric determination of ammonia can be carried out either by the Nessler’* or the Berthelot reactionI or by using pH indicators.” However, special precautions must be taken when these methods are applied to coloured or turbid sample solutions. Few chemiluminometric methods have been established for the determination of ammonia. Ammonia reacts with hypochlorite to form monochloramine in alkaline solution. Thus, the intensity of chemiluminescence (CL) generated during the reaction of luminol with hypochlorite decreases.14 Ammonia has also been determined by oxidative pyrolysis and subsequent measurement of the CL intensity of excited N02.” However, a direct CL method for the determination of ammonia or ammonium ion has not been traced in the literature. This work involves the development and optimization of a method for the analysis of *To whom correspondence should be addressed.
ammonium ion in fertilizers, based on a new CL reaction between N-bromosuccinimide (NBS) and ammonium ion in alkaline medium. During the development of the method, it was found that a number of fluorescent compounds enhance the CL emission intensity if present in the reaction medium. The effect of these compounds on the emission intensity is investigated. Reduction of interferences by cations on the emission intensity is also reported in this paper. EXPERIMENTAL
Apparatus
A schematic diagram of the continuous-flow CL analyser is shown in Fig. 1. It consisted of two basic units, the detector housing and the flow-through system. The detector housing included a coiled glass flow cell situated in front of the photomultiplier tube (PMT). The cell consisted of 3.5 turns of glass tubing (i.d. 2 mm) and its total height was 22 mm. The coil volume was 300 ,ul. The distance of the coil from the PMT was 2 mm for greatest sensitivity. The coil was backed by a mirror for maximum light collection by the PMT. High voltage (- 720 V) was supplied to the PMT (EM1 9783R, S-5 response) by two Heath Universal Power Supplies (O-500 V) connected in series. The output of the PMT was connected to an operational amplifier (RCA CA 3140) which served as a current-tovoltage converter (I-V). Damping was provided
1245
STERGIOS A. HALVATZIS and MEROPIM. TIMOIXEOU-POTAMIA
1246 Sampler
Pump ml min-l 0
GJ
Sample
3.90
NBS
2.90
PMT
0
waste
1
l/V
?!L
Recorder
Hi&h voltr&a
Fig. 1. Schematic diagram of the continuous-flow CL analyser (not to scale).
by inserting an RC circuit between the output of the I-V converter and the recorder. The output of the CL analyser was recorded with a Knauer (Model 73341) recorder. The solutions of reactants were supplied by a Technicon Proportioning Pump III and were mixed at a Y-junction, 20 mm before entering the flow cell. The final solution was carried into the flow cell by a Tygon tube with an i.d. of 2 mm. Samples were supplied to the manifold by a Technicon Sampler II with a 40-sample capacity. Reagents
All solutions were prepared from analyticalreagent grade materials with de-ionized, distilled water. solution N - Bromosuccinimide stock (O.OSOOlw)was prepared daily by dissolving 2.225 g of NBS (Serva) in water, transferring the solution into a volumetric flask and diluting to 250 ml with water. Dichlorofluorescein stock solution (O.OOlOM) was prepared daily by dissolving 0.200 g of dichlorofluorescein (Fluka) in 0.10M sodium hydroxide solution, transferring the solution into a volumetric flask and diluting to 500 ml with the same alkaline solution. Ammonium chloride stock solution (0.100M) (1800 pg/ml of ammonium ion) was prepared by dissolving 2.675 g of ammonium chloride (Merck) in water, transferring the solution into a calibrated flask and diluting to 500 ml with water. More dilute solutions were prepared daily by the minimum number of dilution steps possible. All other common laboratory chemicals were of the best grade available and were used without further purification.
PROCEDURES
Measurement procedure
The instrument was set at the optimized conditions shown in Fig. 1, but the sampling needle was kept in the “wash” position until the baseline on the recorder had been established. The sampler was adjusted to allow analyte or standard solution and washing water to enter the manifold at equal time intervals and at a rate of 106 solutions/hr. The sampler was then activated and the analysis proceeded automatically. A calibration graph was constructed by plotting the emission intensity [Z(mV)] vs. concentration of ammonium ion [C&g/ml)] in standard solutions and the ammonium content of the sample solution was determined. A standard solution after every 12 sample solutions was included. Sample preparation
Ten millilitres of 1.OM sodium hydroxide solution and 10.00 ml of O.OOlOM dichlorofluorescein solution were transferred into a 100 ml volumetric flask with the appropriate volume of stock ammonium ion solution and were diluted to volume with water. For the preparation of fertilizer stock solutions, an amount about 10 g of fertilizer was weighed accurately, transferred into a 1 1 volumetric flask and diluted to volume with water. The mixture was sonicated for 10 min to aid dissolution and then filtered. An appropriate volume of the filtrate was diluted further with water so that the concentration of ammonium ion in the final solution was within the working range (0.0540-5.40 pg/ml). The final solution should also contain 0.10M sodium hydroxide and 1.0 x 10p4M dichlorofluorescein.
1247
Determination of ammonium ion in fertilizers
excite coexisting fluorophores, ofluorescein. A series of experiments were establish the optimum analytical the CL oxidation of ammonium
RESULTS AND DISCUSSION
Chemihuninescent reactions that occur by the action of oxidants containing positively charged bromine atoms are restricted to hypobromiter6 and 1,3-dibromo-5,5-dimethyl-hydantoin.” NBromosuccinimide belongs to the same group of compounds, it is more stable than hypobromite and has been used extensively as a brominating and oxidizing agent. I8Recently, the CL intensity generated during the oxidation of isoniazid by NBS has been found to increase when ammonia is present.lg This observation was attributed to the CL oxidation of ammonia to nitrogen by NBS. The reaction which occurs is?”
like dichlorconducted to conditions for ions by NBS.
Eflect of flow-rate Figure 2 shows the effect of flow-rate of NBS on the emission intensity from 18.0 and 54.0 pg/ml of ammonium ion. At flow-rates of oxidant 22.90 ml/min, the emission intensity is independent of NBS flow-rate. This was expected since the concentration of NBS is at least four times higher than the stoichiometric and the reaction is therefore, pseudofirst-order with respect to ammonium ions. The chosen values for reagent and sample flow-rates were 2.90 and 3.90 ml/min, respectively. The manifold configuration and the optimized flow-rates allow the constituents of the final solution to react for about 0.6 set before entering into the cell. The sampling and wash times of the sampler were chosen to give sharp and smooth peaks. Under these experimental conditions, the response
NH:+OH--+NH3+Hz0 3NBS + 2NH, + 30H+3NHS + Nz + 3Br- + 3Hz0 NHS: succinimide. The reaction probably belongs to the group of CL redox reactions which generate nitrogen. *’ Nitrogen is probably produced in an excited state,** and has the ability to chemi-
100 t
0
1
I
I
I
I
1
2
3
4
Flow-r&e,
ml mfn-l
Fig. 2. Effect of the flow-rate of 0.15OMNBS in 0.5OM sodium hydroxide on the CL intensity, from 18.0 (broken line) and 54.0 (solid line) pg/ml of ammonium ion supplied to the manifold at (1) 2.50, (2) 2.90, (3) 3.40 and (4) 3.90 ml/min.
STERGI~S A. HALVATZIS and MEROPIM. TIMOTHEOU-POTAMIA
1248
was equivalent to 95% of that from the steady state. E$ect of NBS and alkali concentrations The oxidation of ammonium ions by NBS is accompanied by radiation only when the reaction is carried out in alkaline medium. The effect of NBS concentration on 18.0 and 54.0 pg/ml of ammonium ions at various sodium hydroxide concentrations (range 0.010-2.0 M) is shown in Fig. 3. Concentrations of 0.0150M for NBS and 0.30M for sodium hydroxide were determined to be optimal. A decrease in light intensity at higher concentrations of NBS was observed probably due to kinetic reasons. The rate increases and the reaction proceeds to completion before entrance of the solution into the measuring cell. The same reason explains the decrease of the signal at concentrations of sodium hydroxide > 0.5iU. Eflect of surfactants and fluorescent compounds
The efficiency of some CL reactions increases by using surfactants. 23 Their action alters favourably chemical pathways and reaction rates= and, therefore, the sensitivity of the analytical measurement is greatly improved. However, typical surfactants did not show such
0
an effect on the CL reaction examined. Each surfactant tested, when present in concentrations higher than the critical micellar concentration (CMC), caused severe reduction in response due to suppression of the CL reaction and quenching of the emission. The effect of 3-cyclohexylamino-propanesulphonic acid (CAPS), a well known CL sensitizer,*’ on the emission intensity, was also investigated. Concentrations of CAPS > 1.0 x 10m4M reduce severely the emission intensity from ammonium ion. Fluorescent compounds, such as rhodamine B 26 fluorescein and riboflavin,** have been ukd as sensitizers in CL procedures. Their action is mainly due to energy transfer.29 In this work, fluorescent compounds which belong to the triphenylmethane dyes containing an “0x0 bridge” were examined. The oxidation of these dyes was accompanied by light emission.M The chemiluminescence is due to the emission from singlet oxygen and intermolecular energy transfer.31 However, a mixed solution of ammonium ion and a fluorescent compound generates emission with intensity higher than the sum of the intensities from each component of the mixture. This observation is due to energy transfer from the excited states of nitrogen (produced by the
1
2
[NaOH] , Y Fig. 3. Effect of concentration of sodium hydroxide on the CL intensity from 18.0 (broken line) and 54.0 (solid line) cg/ml of ammonium ion with (1) 0.0050, (2) 0.0150 and (3) 0.050M NBS.
Determination of ammonium ion in fertilizers Table 1. Effect of fluorescent compounds on the CL emission from 18.Opg/ml of ammonium ion Ammonium ion @g/ml) 0 18.0 Compound
Concentration (W
None
Emission intensity (m V) *
17.2
Bhodamine B
1.0 x 1.0 x 1.0 x 1.0 x
10-e 10-S IO-4 IO-’
* * * 1.2
17.2 20.5 38.4 45.9
Fluorescein
1.0 x 1.0 x 1.0 x 1.0 x
1O-6 10-j 10-d 10-3
2.3 5.5 10.8 21.2
20.6 47.3 206 411
Dichlorofluorescein
1.0 x 1.0 x 1.0 x 1.0 x
lo+ 1O-5 10-4 10-r
2.6 8.0 18.4 59.0
34.7 224 1420 624
*Not detectable.
reaction of ammonium ion with NBS) to the emitting species produced by the reaction of fluorescent compound with NBS which caused
1249
an increase in the CL efficiency. Table 1 shows that the most enhanced emission was obtained when 1.0 x 10m4Mdichlorofluorescein was present in the analyte solution. As the addition of dichlorofluorescein allows the determination of very low concentrations of ammonium ions, the effect of sodium hydroxide on the emission intensity was re-investigated (Fig. 4). When dichlorofluorescein was also added to the ammonium ion solution the optimum was O.lOM sodium hydroxide, while the optimum values of NBS concentration and flow rates of reagents remain unaffected. Analytical parameters A typical recording for a series of ammonium ion standards obtained by the proposed method is shown in Fig. 5. The calibration graph [I(mV) vs. concentration, C @g/ml)] was linear in the range 0.054M.540pg/ml of ammonium ion I = (11.9 f 0.38) + (75.4 + 1.3)C; r = 0.9992,
(n = 7)
Fig. 4. Effect of concentration of sodium hydroxide on the CL intensity from 0.180 (broken line) and 0.900 (solid line) rg/ml of ammonium ion @Xhlorofluorescein] = 1.0 x 10w4M and [NBS] = O.OlSOM).
1250
STERGIOS
A.
and MEROPIM. TIMOTHEOU-POTAMIA
HALVATZIS
3.60
f
80 mV I
1.80
Y
5min
Fig. 5. Typical recording output for the NBS-ammonium ion reaction under the recommended conditions (the numbers above each set of peaks are micrograms per millilitre of ammonium ion).
and in the range 0.540-5.4Opg/ml monium ion
of am-
I = (-49.6
f 11) + (225 + 3.7)C;
r = 0.999,
(n = 11).
The change of slope of the calibration graph was attributed to the increase of energy transfer yield from the lower to the higher ammonium ion concentration range. The detection limit (blank + three times its standard deviation32) was 0.032 pg/ml of ammonium ion. Repeatability was measured with standard solutions. The relative standard deviations for 10 measurements were 1.6 and 0.4% for 0.540 and 3.60 pg/ml, respectively. Aqueous solutions of ammonium ion (0.090&5.40 pg/ml) were analysed as samples of unknown concen-
tration with a mean relative error of 1.3% (range O-3.5%). Interference studies Interferences from anions were investigated by recovering 1.80 pg/ml of ammonium ions in the presence of 180.0, 18.0 and 1.80 pug/ml of anion. The results are shown in Table 2. No CL emission was observed from pure solutions of the anions except sulphide, which therefore interferes severely. No effect was observed from nitrate and phosphate which are major constituents of solid fertilizers. Hexacyanoferrate(II), arsenite, nitrite and sulphide interfere since they react with NBS and reduce its concentration. Interferences from cations were investigated by the same procedure followed for anions.
Determination
of ammonium ion in fertilizers
1251
Table 2. Analyticalrecoveryof 1.80rg/ml of ammonium When citrate was present in the analyte solion from solutions which contain various anions ution, the detection limit was 0.450 pg/ml of Concentration ratio Recovery ammonium ion and the relative standard devi(anion : ammonium ion) Anion (%) (n = 3) ations for 10 measurements were 1.8 and 0.5% PO:100 100.4 for 0.600 and 4.50 pg/ml, respectively. NO, 100 100.6 The results of the recovery studies for cations Cq100 99.6 so:100 100.1 are shown in Table 4. Manganese(I1) was the Cl100 100.8 most severe interferant of all the cations examBr100 101.6 ined. Cobalt(I1) and chromium(II1) increase the I100 96.5 c,o:100 103.3 intensity, probably due to a catalytic effect.33*34 AsO, 100 88.4 The unusual effect of copper(I1) is probably due 10 96.6 to the absorption of radiation by the coloured NO,100 45.6 10 75.6 solution and the catalytic effect of cation on the I 90.3 CL reaction. [Fe(CWJ100 0 Since many fertilizers contain urea, the effect 10 16.0 1 19.8 of this compound on the emission intensity S2100 34.2 was also investigated. The recovery results of 10 76.7 1.80 pg/ml of ammonium ions from solutions 1 100.3 with 18.0, 1.80 and 0.180 pg/ml of urea were 153.1, 110.4 and 100.3% (n = 3), respectively. The increase in the CL intensity is attributed to However, most cations form insoluble compounds in alkaline medium. Formation of preTable 4. Analyticalrecoveryof 1.80pg/ml of ammonium cipitates can be avoided by using complexing ion from solutions which contain various cations and agents. Common complexing agents were found O.OlOM sodiumcitrate to decrease the emission intensity from amConcentrationratio Recovery monium ions due to quenching effect (Table 3). Cation (cation:ammonium) (%) (tl = 3) The least severe reduction in response is caused 100 104.0 Mg2+ by citrate which was used at O.OlOM. This Ni2+ 100 80.4 concentration is in adequate excess for complex10 103.6 ing the cations tested. The equation of the CU2f 100 90.0 calibration graph, using citrate in standard sol10 114.4 1 123.2 utions, obtained in the range 0.540-5.40 pg/ml Cr’+ 100 390.5 of ammonium ion was: 10 227.3 1 210.1 Z = (- 102 + 12) + (227 + 4.O)C; r = 0.999,
(n = 9).
Table 3. Effect of some common complexing agents on the CL emission from 1.80 pg/ml of ammonium ion (emission intensity from pure ammonium ion = 100) Compound
Concentration
(M)
Relative emission intensity (%)
Mn*+
100,lO 1
Fe’+
100 10
* 92.1 * 96.8
A13+
100 10
91.5 96.7
Pb2+
100 10 1
83.5 93.5 99.3
EGTA*
0.0010 0.0100
37.5 3.5
Zn2+
DCTAt
0.0010 0.0100
59.7 8.5
100 10
93.8 104.3
Gas+
EDTA#
0.0010 0.0100
59.7 9.0
100 10
110.5 100.5
Citrate
0.0010 0.0100 0.1000
97.2 91.7 50.7
lEthyleneglycol - bis(2 - aminoethylether) - N,N,N’,N’ - tetraacetic acid. tl,2-Diaminocyclohexane-N,N,N’,N’-tetraacetic fEthylenediamino-N,N,N’,N’-tetraacetic acid.
acid.
Ba2+
100
102.0
co*+
100 10 1
229.3 128.1 103.4
Cd2+
100 10
100.1
l
*Precipitate formed after mixing of ammonium and metal ion solution.
1252
SIERGIOS
A. HALVATZBand MEROPIM. TIMOTHEOU-POTAMIA
ammonia produced by hydrolysis of urea in alkaline medium. The method can only be ap plied to samples that also contain urea at concentrations lower than that of ammonium ion. Accuracy
The accuracy of the proposed continuousflow CL method was examined by performing recovery experiments on solutions prepared from commercial solid and liquid fertilizers. Table 5 summarizes the results of these studies. O.OlOM sodium citrate was added to all solutions used in the analysis of samples which contained cations, in order to avoid formation of precipitates. Average recoveries were 100.6% (97.0-106.1%) and 110.9% (104.6-l 19.7%) for solid and liquid samples, respectively. High recoveries of ammonium ion from liquid samples might be related to the effect of cations, such as copper(I1). The proposed method was
also evaluated by analysing commercial samples and the results are compared with the values determined by the standard method.35 A satisfactory agreement between the results was obtained (Table 6), with a mean relative difference of 1.3 and 4.2% for solid and liquid samples, respectively. CONCLUSIONS
The proposed automated method is the first direct chemiluminometric method developed for ammonium ions. Dilution is the only sample pre-treatment required and the detection limit, sensitivity and selectivity compare very well with existing analytical methods for ammonium ion. The results are repeatable and show that the method can be applied to the determination of ammonium ion in fertilizers. Urea is the only severe interferent when present at concentration ratios 3 1. The method has no interferences
Table 5. Recovery experiments for ammonium ion added to sample solution of commercial fertilizers Ammonium ion @g/ml) Sample
Initially present
Added
Recovered
Recovery, (%) (a = 3)
Solid fertilizer No 1
0.875
0.810 1.800 2.700
0.834 1.746 2.658
103.0 97.0 98.4
No 6
0.854
0.810 1.800 2.700
0.823 1.800 2.701
101.6 100.0 100.0
No 13
0.910
0.810 1.800 2.700
0.826 1.791 2.654
102.0 99.5 98.3
No 21
0.907
0.810 1.800 2.700
0.852 1.790 2.704
103.1 99.4 100.1
No 22
0.904
0.810 1.800 2.700
0.838 1.738 2.708
103.5 99.1 100.3
Fleran*
1.012
0.540 0.900 1.800
0.526 0.923 1.909 Mean:
97.4 102.6 106.1 100.6
Liquid fertilizer Viofyt*
1.045
0.540 0.900 1.800
0.611 1.077 2.112
113.1 119.7 117.3
Algoflash*
1.052
0.540 0.900 1.800
0.569 0.947 1.893
105.4 105.2 105.2
Anthin*
1.212
0.540 0.900 1.800
0.565 0.982 2.125 Mean:
104.6 109.1 118.1 110.9
*The solutions contain O.OlOM sodium citrate.
1253
Determination of ammonium ion in fertilizers Table 6. Determination
of ammonium-nitrogen in commercial fertilizers with the proposed method and the standard method3s Ammonium-nitrogen
Sample
Composition (%N-%P,O,-%K,O)
Solid No 1 No 2 No 3 No 4
Proposed method (&SD)+
(%)
Standard method
Relative difference (%)
20-10-0
18.85 f 18.74 f 18.90 f 19.08 * 18.72 +
0.08 0.16 0.02 0.08 0.10
18.94 19.07 19.04 19.18 19.05
-0.5 -1.7 -0.7 -0.5 -1.7
6 I 8 9 10 11 12
16-20-O
15.48 f 0.05 15.47 + 0 15.56 & 0.19 15.63 +_0.05 15.53 + 0.04 15.44 If:0.06 15.22 f 0.03
15.57 15.92 15.48 15.53 15.46 15.50 15.57
-0.6 -2.8 +0.5 +0.6 +0.5 -0.4 -2.2
No 13 No 14 No 15 No 16 No 17 No 18 No 19 No 20
11-1515
11.32&O 10.78 + 0.10 11.33 10.04 11.01 *0.06 11.05 + 0.02 11.14*0.07 10.98 f 0.06 11.03 kO.02
11.21 10.93 11.00 11.04 10.79 11.15 10.89 10.79
+1.0 -1.4 +3.0 -0.3 -l-2.4 -0.1 +0.8 +2.2
No 21 No 22 Flerant
8-20-20
8.58 f 0.02
8.52 4.63 20.46 Mean:
+0.7 +0.4 -3.9 1.3
4.83 1.98 15.12 Mean:
+5.2 +3.5 +3.8 4.2
No 5
No No No No No No No
Liquid Viofjttb Algoflasht Anthintd
2El5
5-10-5 l_
19.67 4.65 + f 0.04 0.25
5.08 f 0.03 2.05 f 0.01 15.70 f 0
*Standard deviation (n = 3). tThe standard and sample solutions contain O.OlOM sodium citrate because these samples contain (a) Mg, Mn, B, Pb; (b) Fe, Cu, Zn, Mg, B, Mn, Ca, S; (c) Fe, Cu, Zn, Pb, B; (d) Fe, Cu, Mn, Mg, B.
from common anions, such as nitrate and phosphate and the interferences from cations have been significantly reduced or eliminated. Acknowledgements-The authors are grateful to Prof. A. C. Calokerinos for helpful discussions. One of the authors (S. A. H.) thanks the University of Athens for fmancial support. REFERENCES 1. T. Aoki, S. Uemura and M. Munemori, Anal. Gem., 1983, 55, 1620. 2. Z. Genfa and P. K. Dasgupta, Anal. Chem., 1989, 61, 408. 3. R. Belcher, S. L. Bogdanski, A. C. Calokerinos and A. Townshend, Analyst, 1981, 106, 625. 4. V. C. Anigbogu, M. L. Dietz and A. Syty, Anal. Chem., 1983, 55, 535. 5. K. Fukushi and K. Hiiro, Talanta, 1988, 35, 799. 6. S. Alegret, J. Alonso, J. Bartroli and E. MartinezFabregas, Analyst, 1989, 114, 1443. 7. F. E. Friedl, Anal. Biochem., 1972, 48, 300.
8. C. Pasquini and Lourival Cardoso De Faria, Anal. Chim. Acta, 1987, 193, 19. 9. I. Sajo and B. Sipos, Talanta, 1972, 19, 669. 10. W. E. Van Der Linden, Anal. Chzin. Acta, 1983, 151, 359. 11. R. Nakata, T. Kawamura, H. Sakashita and A. Nitta, Anal. Chim. Acta, 1988, 2M, 81. 12. H. Bergamin, B. F. Reis, A. 0. Jacintho and E. A. G. Zagatto, Anal. Chim. Acta, 1980, 117, 81. 13. C. Pasquini and W. A. Oliviera, Anul. Chem., 1985,57, 2515. 14. P. R. Kraus and S. R. Crouch, Anal. L&t., 1987, Zo, 183. 15. K. Gerth, Anal. Biochem., 1985, 144,432. 16. J. Teckentrup and D. Klockow, Talanta, 1981, 2% 653. 17. A. T. Pilipenlo, 0. V. Zui and A. V. Terletskaya, J. Anal. Clrem., U.S.S.R., 1986, 41, 560. 18. N. K. Mathour and C. K. Narang, The Determination of Organic Cornpour& with N-Bromosuccinimide and Allied Reagents. Academic Press, London, 1975. 19. S. A. Halvatzis, M. M. Timotheou-Potamia and A. C. Calokerinos, Adyst, 1990, 115, 1229.
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STERGIOSA. HALVATZIS and MEROPIM. TIMOTHWIU-POTAMIA
20. G. W. Stevenson and J. M. Luck, J. Biol. Chem., 1961, 236, 715. 21. F. McCapra, Prog. Org. Chem., 1973, 8, 231. 22. H. Jurgensen and J. D. Winefordner, Talanta, 1984,31, 777. 23. T. E. Riehl, C. L. Malehom and W. L. Hinze, Analyst, 1986, 111,931. 24. E. Pelirzetti and E. Pramauro, Anal. Chim. Acta, 1985, 169,l. 25. I. I. Koukli, E. G. Sarantonis and A. C. Calokerinos, Anal. L.&t., 1990, 23, 1167. 26. J. S. Lancaster, P. J. Worsfold and A. Lynes, Analyst, 1989, 114, 1659. 27. M. Yamada and S. Suzuki, Anal. Chim. Acta, 1987,193, 337.
28. 8. A. Al-Tam&, A. Townshend and A. R. Wheatley, Analyst, 1987, 112, 883. 29. M. Yamada, T. Nakada and S. Suzuki, Anal. Chim. Acta, 1983, 147, 401. 30. G. N. Chen and C. S. Huang, Talanta, 1988, 35, 625. 31. D. Slawinska and J. Slawinski, Anal. Chem., 1975, 47, 2101. 32. J. C. Miller and J. N. Miller, Statistics for Analytical Chemistry. Hot-wood, Chichester, 1986. 33. I. M. A. Shakir and A. T. Faizullah, Analyst, 1989, 114, 951. 34. V. I. Rigin and A. I. Blokhin, J. Anal. Chem. U.S.S.R., 1977, 32, 246. 35. International Organization for Standardization, International Standard IS0 53 14-1981.
0039-9140/93 $6.00 + 0.00
Vol. 40, No. 8, pp. 1245-1254, 1993
Printedin Great Britain. All rights remwd
copyright0 1993 Pcrgamon Fvcss Ltd
CONTINUOUS-FL.OW
CHEMILUMINOMETRIC
DETERMINATION OF AMMONIUM IN FERTILIZERS
ION
STERGIOSA. HALVATZISand MJZROPI M. TIMOTHEOU-POTAMIA* Laboratory of Analytical Chemistry, University of Athens, Panepistimiopolis, 157 71 Athens, Greece (Received 3 Augusf 1992. Revised 8 January 1993. Accepted 25 January 1993) Summary-A simple continuous-flow chemiluminometric method for the determination of 0.0540-5.4O~&ml of ammonium ion is described. The method is based on the chemiltinescence generated during the oxidation of ammonium ion by N-bromosuccinimide in alkaline medium. The emission intensity is greatly enhanced if dichlorofluorescein is also present in the reaction solution. The analysis is automated, requires no sample pre-treatment and solutions can be analysed at a rate of 106 solution@ with a relative error of about 2.5%. The method was applied satisfactorily to the determination of ammonium ion in solid and liquid fertilizers.
A wide variety of methods and techniques are available for the determination of ammonium ion. Spectrofluorimetry,‘J molecular emission cavity analysis,3 gas phase molecular absorption spectrometry,4 capillary isotachophoreGs,s ionselective electrodes,‘j conductometry,‘T8 and thermometric titrimetryg are only some examples of the techniques available for the determination of ammonia and ammonium ions. Flow injection systems with gas diffusion or ion exchange modules and spectrophotometric detection are described in the literature.‘“‘* The spectrophotometric determination of ammonia can be carried out either by the Nessler’* or the Berthelot reactionI or by using pH indicators.” However, special precautions must be taken when these methods are applied to coloured or turbid sample solutions. Few chemiluminometric methods have been established for the determination of ammonia. Ammonia reacts with hypochlorite to form monochloramine in alkaline solution. Thus, the intensity of chemiluminescence (CL) generated during the reaction of luminol with hypochlorite decreases.14 Ammonia has also been determined by oxidative pyrolysis and subsequent measurement of the CL intensity of excited N02.” However, a direct CL method for the determination of ammonia or ammonium ion has not been traced in the literature. This work involves the development and optimization of a method for the analysis of *To whom correspondence should be addressed.
ammonium ion in fertilizers, based on a new CL reaction between N-bromosuccinimide (NBS) and ammonium ion in alkaline medium. During the development of the method, it was found that a number of fluorescent compounds enhance the CL emission intensity if present in the reaction medium. The effect of these compounds on the emission intensity is investigated. Reduction of interferences by cations on the emission intensity is also reported in this paper. EXPERIMENTAL
Apparatus
A schematic diagram of the continuous-flow CL analyser is shown in Fig. 1. It consisted of two basic units, the detector housing and the flow-through system. The detector housing included a coiled glass flow cell situated in front of the photomultiplier tube (PMT). The cell consisted of 3.5 turns of glass tubing (i.d. 2 mm) and its total height was 22 mm. The coil volume was 300 ,ul. The distance of the coil from the PMT was 2 mm for greatest sensitivity. The coil was backed by a mirror for maximum light collection by the PMT. High voltage (- 720 V) was supplied to the PMT (EM1 9783R, S-5 response) by two Heath Universal Power Supplies (O-500 V) connected in series. The output of the PMT was connected to an operational amplifier (RCA CA 3140) which served as a current-tovoltage converter (I-V). Damping was provided
1245
STERGIOS A. HALVATZIS and MEROPIM. TIMOIXEOU-POTAMIA
1246 Sampler
Pump ml min-l 0
GJ
Sample
3.90
NBS
2.90
PMT
0
waste
1
l/V
?!L
Recorder
Hi&h voltr&a
Fig. 1. Schematic diagram of the continuous-flow CL analyser (not to scale).
by inserting an RC circuit between the output of the I-V converter and the recorder. The output of the CL analyser was recorded with a Knauer (Model 73341) recorder. The solutions of reactants were supplied by a Technicon Proportioning Pump III and were mixed at a Y-junction, 20 mm before entering the flow cell. The final solution was carried into the flow cell by a Tygon tube with an i.d. of 2 mm. Samples were supplied to the manifold by a Technicon Sampler II with a 40-sample capacity. Reagents
All solutions were prepared from analyticalreagent grade materials with de-ionized, distilled water. solution N - Bromosuccinimide stock (O.OSOOlw)was prepared daily by dissolving 2.225 g of NBS (Serva) in water, transferring the solution into a volumetric flask and diluting to 250 ml with water. Dichlorofluorescein stock solution (O.OOlOM) was prepared daily by dissolving 0.200 g of dichlorofluorescein (Fluka) in 0.10M sodium hydroxide solution, transferring the solution into a volumetric flask and diluting to 500 ml with the same alkaline solution. Ammonium chloride stock solution (0.100M) (1800 pg/ml of ammonium ion) was prepared by dissolving 2.675 g of ammonium chloride (Merck) in water, transferring the solution into a calibrated flask and diluting to 500 ml with water. More dilute solutions were prepared daily by the minimum number of dilution steps possible. All other common laboratory chemicals were of the best grade available and were used without further purification.
PROCEDURES
Measurement procedure
The instrument was set at the optimized conditions shown in Fig. 1, but the sampling needle was kept in the “wash” position until the baseline on the recorder had been established. The sampler was adjusted to allow analyte or standard solution and washing water to enter the manifold at equal time intervals and at a rate of 106 solutions/hr. The sampler was then activated and the analysis proceeded automatically. A calibration graph was constructed by plotting the emission intensity [Z(mV)] vs. concentration of ammonium ion [C&g/ml)] in standard solutions and the ammonium content of the sample solution was determined. A standard solution after every 12 sample solutions was included. Sample preparation
Ten millilitres of 1.OM sodium hydroxide solution and 10.00 ml of O.OOlOM dichlorofluorescein solution were transferred into a 100 ml volumetric flask with the appropriate volume of stock ammonium ion solution and were diluted to volume with water. For the preparation of fertilizer stock solutions, an amount about 10 g of fertilizer was weighed accurately, transferred into a 1 1 volumetric flask and diluted to volume with water. The mixture was sonicated for 10 min to aid dissolution and then filtered. An appropriate volume of the filtrate was diluted further with water so that the concentration of ammonium ion in the final solution was within the working range (0.0540-5.40 pg/ml). The final solution should also contain 0.10M sodium hydroxide and 1.0 x 10p4M dichlorofluorescein.
1247
Determination of ammonium ion in fertilizers
excite coexisting fluorophores, ofluorescein. A series of experiments were establish the optimum analytical the CL oxidation of ammonium
RESULTS AND DISCUSSION
Chemihuninescent reactions that occur by the action of oxidants containing positively charged bromine atoms are restricted to hypobromiter6 and 1,3-dibromo-5,5-dimethyl-hydantoin.” NBromosuccinimide belongs to the same group of compounds, it is more stable than hypobromite and has been used extensively as a brominating and oxidizing agent. I8Recently, the CL intensity generated during the oxidation of isoniazid by NBS has been found to increase when ammonia is present.lg This observation was attributed to the CL oxidation of ammonia to nitrogen by NBS. The reaction which occurs is?”
like dichlorconducted to conditions for ions by NBS.
Eflect of flow-rate Figure 2 shows the effect of flow-rate of NBS on the emission intensity from 18.0 and 54.0 pg/ml of ammonium ion. At flow-rates of oxidant 22.90 ml/min, the emission intensity is independent of NBS flow-rate. This was expected since the concentration of NBS is at least four times higher than the stoichiometric and the reaction is therefore, pseudofirst-order with respect to ammonium ions. The chosen values for reagent and sample flow-rates were 2.90 and 3.90 ml/min, respectively. The manifold configuration and the optimized flow-rates allow the constituents of the final solution to react for about 0.6 set before entering into the cell. The sampling and wash times of the sampler were chosen to give sharp and smooth peaks. Under these experimental conditions, the response
NH:+OH--+NH3+Hz0 3NBS + 2NH, + 30H+3NHS + Nz + 3Br- + 3Hz0 NHS: succinimide. The reaction probably belongs to the group of CL redox reactions which generate nitrogen. *’ Nitrogen is probably produced in an excited state,** and has the ability to chemi-
100 t
0
1
I
I
I
I
1
2
3
4
Flow-r&e,
ml mfn-l
Fig. 2. Effect of the flow-rate of 0.15OMNBS in 0.5OM sodium hydroxide on the CL intensity, from 18.0 (broken line) and 54.0 (solid line) pg/ml of ammonium ion supplied to the manifold at (1) 2.50, (2) 2.90, (3) 3.40 and (4) 3.90 ml/min.
STERGI~S A. HALVATZIS and MEROPIM. TIMOTHEOU-POTAMIA
1248
was equivalent to 95% of that from the steady state. E$ect of NBS and alkali concentrations The oxidation of ammonium ions by NBS is accompanied by radiation only when the reaction is carried out in alkaline medium. The effect of NBS concentration on 18.0 and 54.0 pg/ml of ammonium ions at various sodium hydroxide concentrations (range 0.010-2.0 M) is shown in Fig. 3. Concentrations of 0.0150M for NBS and 0.30M for sodium hydroxide were determined to be optimal. A decrease in light intensity at higher concentrations of NBS was observed probably due to kinetic reasons. The rate increases and the reaction proceeds to completion before entrance of the solution into the measuring cell. The same reason explains the decrease of the signal at concentrations of sodium hydroxide > 0.5iU. Eflect of surfactants and fluorescent compounds
The efficiency of some CL reactions increases by using surfactants. 23 Their action alters favourably chemical pathways and reaction rates= and, therefore, the sensitivity of the analytical measurement is greatly improved. However, typical surfactants did not show such
0
an effect on the CL reaction examined. Each surfactant tested, when present in concentrations higher than the critical micellar concentration (CMC), caused severe reduction in response due to suppression of the CL reaction and quenching of the emission. The effect of 3-cyclohexylamino-propanesulphonic acid (CAPS), a well known CL sensitizer,*’ on the emission intensity, was also investigated. Concentrations of CAPS > 1.0 x 10m4M reduce severely the emission intensity from ammonium ion. Fluorescent compounds, such as rhodamine B 26 fluorescein and riboflavin,** have been ukd as sensitizers in CL procedures. Their action is mainly due to energy transfer.29 In this work, fluorescent compounds which belong to the triphenylmethane dyes containing an “0x0 bridge” were examined. The oxidation of these dyes was accompanied by light emission.M The chemiluminescence is due to the emission from singlet oxygen and intermolecular energy transfer.31 However, a mixed solution of ammonium ion and a fluorescent compound generates emission with intensity higher than the sum of the intensities from each component of the mixture. This observation is due to energy transfer from the excited states of nitrogen (produced by the
1
2
[NaOH] , Y Fig. 3. Effect of concentration of sodium hydroxide on the CL intensity from 18.0 (broken line) and 54.0 (solid line) cg/ml of ammonium ion with (1) 0.0050, (2) 0.0150 and (3) 0.050M NBS.
Determination of ammonium ion in fertilizers Table 1. Effect of fluorescent compounds on the CL emission from 18.Opg/ml of ammonium ion Ammonium ion @g/ml) 0 18.0 Compound
Concentration (W
None
Emission intensity (m V) *
17.2
Bhodamine B
1.0 x 1.0 x 1.0 x 1.0 x
10-e 10-S IO-4 IO-’
* * * 1.2
17.2 20.5 38.4 45.9
Fluorescein
1.0 x 1.0 x 1.0 x 1.0 x
1O-6 10-j 10-d 10-3
2.3 5.5 10.8 21.2
20.6 47.3 206 411
Dichlorofluorescein
1.0 x 1.0 x 1.0 x 1.0 x
lo+ 1O-5 10-4 10-r
2.6 8.0 18.4 59.0
34.7 224 1420 624
*Not detectable.
reaction of ammonium ion with NBS) to the emitting species produced by the reaction of fluorescent compound with NBS which caused
1249
an increase in the CL efficiency. Table 1 shows that the most enhanced emission was obtained when 1.0 x 10m4Mdichlorofluorescein was present in the analyte solution. As the addition of dichlorofluorescein allows the determination of very low concentrations of ammonium ions, the effect of sodium hydroxide on the emission intensity was re-investigated (Fig. 4). When dichlorofluorescein was also added to the ammonium ion solution the optimum was O.lOM sodium hydroxide, while the optimum values of NBS concentration and flow rates of reagents remain unaffected. Analytical parameters A typical recording for a series of ammonium ion standards obtained by the proposed method is shown in Fig. 5. The calibration graph [I(mV) vs. concentration, C @g/ml)] was linear in the range 0.054M.540pg/ml of ammonium ion I = (11.9 f 0.38) + (75.4 + 1.3)C; r = 0.9992,
(n = 7)
Fig. 4. Effect of concentration of sodium hydroxide on the CL intensity from 0.180 (broken line) and 0.900 (solid line) rg/ml of ammonium ion @Xhlorofluorescein] = 1.0 x 10w4M and [NBS] = O.OlSOM).
1250
STERGIOS
A.
and MEROPIM. TIMOTHEOU-POTAMIA
HALVATZIS
3.60
f
80 mV I
1.80
Y
5min
Fig. 5. Typical recording output for the NBS-ammonium ion reaction under the recommended conditions (the numbers above each set of peaks are micrograms per millilitre of ammonium ion).
and in the range 0.540-5.4Opg/ml monium ion
of am-
I = (-49.6
f 11) + (225 + 3.7)C;
r = 0.999,
(n = 11).
The change of slope of the calibration graph was attributed to the increase of energy transfer yield from the lower to the higher ammonium ion concentration range. The detection limit (blank + three times its standard deviation32) was 0.032 pg/ml of ammonium ion. Repeatability was measured with standard solutions. The relative standard deviations for 10 measurements were 1.6 and 0.4% for 0.540 and 3.60 pg/ml, respectively. Aqueous solutions of ammonium ion (0.090&5.40 pg/ml) were analysed as samples of unknown concen-
tration with a mean relative error of 1.3% (range O-3.5%). Interference studies Interferences from anions were investigated by recovering 1.80 pg/ml of ammonium ions in the presence of 180.0, 18.0 and 1.80 pug/ml of anion. The results are shown in Table 2. No CL emission was observed from pure solutions of the anions except sulphide, which therefore interferes severely. No effect was observed from nitrate and phosphate which are major constituents of solid fertilizers. Hexacyanoferrate(II), arsenite, nitrite and sulphide interfere since they react with NBS and reduce its concentration. Interferences from cations were investigated by the same procedure followed for anions.
Determination
of ammonium ion in fertilizers
1251
Table 2. Analyticalrecoveryof 1.80rg/ml of ammonium When citrate was present in the analyte solion from solutions which contain various anions ution, the detection limit was 0.450 pg/ml of Concentration ratio Recovery ammonium ion and the relative standard devi(anion : ammonium ion) Anion (%) (n = 3) ations for 10 measurements were 1.8 and 0.5% PO:100 100.4 for 0.600 and 4.50 pg/ml, respectively. NO, 100 100.6 The results of the recovery studies for cations Cq100 99.6 so:100 100.1 are shown in Table 4. Manganese(I1) was the Cl100 100.8 most severe interferant of all the cations examBr100 101.6 ined. Cobalt(I1) and chromium(II1) increase the I100 96.5 c,o:100 103.3 intensity, probably due to a catalytic effect.33*34 AsO, 100 88.4 The unusual effect of copper(I1) is probably due 10 96.6 to the absorption of radiation by the coloured NO,100 45.6 10 75.6 solution and the catalytic effect of cation on the I 90.3 CL reaction. [Fe(CWJ100 0 Since many fertilizers contain urea, the effect 10 16.0 1 19.8 of this compound on the emission intensity S2100 34.2 was also investigated. The recovery results of 10 76.7 1.80 pg/ml of ammonium ions from solutions 1 100.3 with 18.0, 1.80 and 0.180 pg/ml of urea were 153.1, 110.4 and 100.3% (n = 3), respectively. The increase in the CL intensity is attributed to However, most cations form insoluble compounds in alkaline medium. Formation of preTable 4. Analyticalrecoveryof 1.80pg/ml of ammonium cipitates can be avoided by using complexing ion from solutions which contain various cations and agents. Common complexing agents were found O.OlOM sodiumcitrate to decrease the emission intensity from amConcentrationratio Recovery monium ions due to quenching effect (Table 3). Cation (cation:ammonium) (%) (tl = 3) The least severe reduction in response is caused 100 104.0 Mg2+ by citrate which was used at O.OlOM. This Ni2+ 100 80.4 concentration is in adequate excess for complex10 103.6 ing the cations tested. The equation of the CU2f 100 90.0 calibration graph, using citrate in standard sol10 114.4 1 123.2 utions, obtained in the range 0.540-5.40 pg/ml Cr’+ 100 390.5 of ammonium ion was: 10 227.3 1 210.1 Z = (- 102 + 12) + (227 + 4.O)C; r = 0.999,
(n = 9).
Table 3. Effect of some common complexing agents on the CL emission from 1.80 pg/ml of ammonium ion (emission intensity from pure ammonium ion = 100) Compound
Concentration
(M)
Relative emission intensity (%)
Mn*+
100,lO 1
Fe’+
100 10
* 92.1 * 96.8
A13+
100 10
91.5 96.7
Pb2+
100 10 1
83.5 93.5 99.3
EGTA*
0.0010 0.0100
37.5 3.5
Zn2+
DCTAt
0.0010 0.0100
59.7 8.5
100 10
93.8 104.3
Gas+
EDTA#
0.0010 0.0100
59.7 9.0
100 10
110.5 100.5
Citrate
0.0010 0.0100 0.1000
97.2 91.7 50.7
lEthyleneglycol - bis(2 - aminoethylether) - N,N,N’,N’ - tetraacetic acid. tl,2-Diaminocyclohexane-N,N,N’,N’-tetraacetic fEthylenediamino-N,N,N’,N’-tetraacetic acid.
acid.
Ba2+
100
102.0
co*+
100 10 1
229.3 128.1 103.4
Cd2+
100 10
100.1
l
*Precipitate formed after mixing of ammonium and metal ion solution.
1252
SIERGIOS
A. HALVATZBand MEROPIM. TIMOTHEOU-POTAMIA
ammonia produced by hydrolysis of urea in alkaline medium. The method can only be ap plied to samples that also contain urea at concentrations lower than that of ammonium ion. Accuracy
The accuracy of the proposed continuousflow CL method was examined by performing recovery experiments on solutions prepared from commercial solid and liquid fertilizers. Table 5 summarizes the results of these studies. O.OlOM sodium citrate was added to all solutions used in the analysis of samples which contained cations, in order to avoid formation of precipitates. Average recoveries were 100.6% (97.0-106.1%) and 110.9% (104.6-l 19.7%) for solid and liquid samples, respectively. High recoveries of ammonium ion from liquid samples might be related to the effect of cations, such as copper(I1). The proposed method was
also evaluated by analysing commercial samples and the results are compared with the values determined by the standard method.35 A satisfactory agreement between the results was obtained (Table 6), with a mean relative difference of 1.3 and 4.2% for solid and liquid samples, respectively. CONCLUSIONS
The proposed automated method is the first direct chemiluminometric method developed for ammonium ions. Dilution is the only sample pre-treatment required and the detection limit, sensitivity and selectivity compare very well with existing analytical methods for ammonium ion. The results are repeatable and show that the method can be applied to the determination of ammonium ion in fertilizers. Urea is the only severe interferent when present at concentration ratios 3 1. The method has no interferences
Table 5. Recovery experiments for ammonium ion added to sample solution of commercial fertilizers Ammonium ion @g/ml) Sample
Initially present
Added
Recovered
Recovery, (%) (a = 3)
Solid fertilizer No 1
0.875
0.810 1.800 2.700
0.834 1.746 2.658
103.0 97.0 98.4
No 6
0.854
0.810 1.800 2.700
0.823 1.800 2.701
101.6 100.0 100.0
No 13
0.910
0.810 1.800 2.700
0.826 1.791 2.654
102.0 99.5 98.3
No 21
0.907
0.810 1.800 2.700
0.852 1.790 2.704
103.1 99.4 100.1
No 22
0.904
0.810 1.800 2.700
0.838 1.738 2.708
103.5 99.1 100.3
Fleran*
1.012
0.540 0.900 1.800
0.526 0.923 1.909 Mean:
97.4 102.6 106.1 100.6
Liquid fertilizer Viofyt*
1.045
0.540 0.900 1.800
0.611 1.077 2.112
113.1 119.7 117.3
Algoflash*
1.052
0.540 0.900 1.800
0.569 0.947 1.893
105.4 105.2 105.2
Anthin*
1.212
0.540 0.900 1.800
0.565 0.982 2.125 Mean:
104.6 109.1 118.1 110.9
*The solutions contain O.OlOM sodium citrate.
1253
Determination of ammonium ion in fertilizers Table 6. Determination
of ammonium-nitrogen in commercial fertilizers with the proposed method and the standard method3s Ammonium-nitrogen
Sample
Composition (%N-%P,O,-%K,O)
Solid No 1 No 2 No 3 No 4
Proposed method (&SD)+
(%)
Standard method
Relative difference (%)
20-10-0
18.85 f 18.74 f 18.90 f 19.08 * 18.72 +
0.08 0.16 0.02 0.08 0.10
18.94 19.07 19.04 19.18 19.05
-0.5 -1.7 -0.7 -0.5 -1.7
6 I 8 9 10 11 12
16-20-O
15.48 f 0.05 15.47 + 0 15.56 & 0.19 15.63 +_0.05 15.53 + 0.04 15.44 If:0.06 15.22 f 0.03
15.57 15.92 15.48 15.53 15.46 15.50 15.57
-0.6 -2.8 +0.5 +0.6 +0.5 -0.4 -2.2
No 13 No 14 No 15 No 16 No 17 No 18 No 19 No 20
11-1515
11.32&O 10.78 + 0.10 11.33 10.04 11.01 *0.06 11.05 + 0.02 11.14*0.07 10.98 f 0.06 11.03 kO.02
11.21 10.93 11.00 11.04 10.79 11.15 10.89 10.79
+1.0 -1.4 +3.0 -0.3 -l-2.4 -0.1 +0.8 +2.2
No 21 No 22 Flerant
8-20-20
8.58 f 0.02
8.52 4.63 20.46 Mean:
+0.7 +0.4 -3.9 1.3
4.83 1.98 15.12 Mean:
+5.2 +3.5 +3.8 4.2
No 5
No No No No No No No
Liquid Viofjttb Algoflasht Anthintd
2El5
5-10-5 l_
19.67 4.65 + f 0.04 0.25
5.08 f 0.03 2.05 f 0.01 15.70 f 0
*Standard deviation (n = 3). tThe standard and sample solutions contain O.OlOM sodium citrate because these samples contain (a) Mg, Mn, B, Pb; (b) Fe, Cu, Zn, Mg, B, Mn, Ca, S; (c) Fe, Cu, Zn, Pb, B; (d) Fe, Cu, Mn, Mg, B.
from common anions, such as nitrate and phosphate and the interferences from cations have been significantly reduced or eliminated. Acknowledgements-The authors are grateful to Prof. A. C. Calokerinos for helpful discussions. One of the authors (S. A. H.) thanks the University of Athens for fmancial support. REFERENCES 1. T. Aoki, S. Uemura and M. Munemori, Anal. Gem., 1983, 55, 1620. 2. Z. Genfa and P. K. Dasgupta, Anal. Chem., 1989, 61, 408. 3. R. Belcher, S. L. Bogdanski, A. C. Calokerinos and A. Townshend, Analyst, 1981, 106, 625. 4. V. C. Anigbogu, M. L. Dietz and A. Syty, Anal. Chem., 1983, 55, 535. 5. K. Fukushi and K. Hiiro, Talanta, 1988, 35, 799. 6. S. Alegret, J. Alonso, J. Bartroli and E. MartinezFabregas, Analyst, 1989, 114, 1443. 7. F. E. Friedl, Anal. Biochem., 1972, 48, 300.
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STERGIOSA. HALVATZIS and MEROPIM. TIMOTHWIU-POTAMIA
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