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Correction of iron interface in the spectrophotometric flow injection catalytic determination of molybdenum in plants
Talanta ELSEVIER
Talanta 42 (1995) 2021-2026
Correction of iron interface in the spectrophotometric flow injection catalytic determination of molybdenum in plants Elma Neide Vasconcelos Martins Carrilho, Francisco Jos6 Krug *, Elias Ayres Guidetti Zagatto Centro de Energia Nuclear na Agricultura, Universidade de S~o Paulo, P.O. Box 96, 13400-970 Piracicaba, Sao Paulo, Brazil
Received 12 April 1995; revised 5 July 1995; accepted 7 July 1995
Abstract
Iron interference in the spectrophotometric catalytic determination of molybdenum based on the iodidehydrogen peroxide reaction can be corrected by using sulphosalicylic acid as masking and color-forming reagent. The catalytic influence of iron ions is circumvented to the extent of about 90% and correction of any remaining iron ions is possible by monitoring the colored iron(III)-salicylate complex at 490 rim. In this way, iron is also determined. With the proposed system, molybdenum can be determined in plant and food digests within the 0-100 I~g Mo 1-t range in the presence of up to 25 mg Fe !-t, at a sampling rate of about 50 determinations h-t. The relative standard deviation of 10 consecutive measurements was estimated as < 2%. Results for samples were comparable with those obtained by graphite furnace atomic absorption spectrometry. In addition, recoveries within the range 94-100% were calculated. Keywords: Iron interference; Molybdenum determination; Sulphosalicylic acid; Flow injection; Plants
1. Introduction
Molybdenum is an essential nutrient for animals and plants and its determination in plant, soil and food materials is o f utmost importance. Nevertheless, only a few well-equipped laboratories accomplish molybdenum determination on a routine basis. In general, molybdenum occurs in low concentrations in plant [1,2] ( 0 . 5 - 3 . 0 m g k g -I) and food materials, which makes the direct determination in the sample digest by simple techniques such as classical spectrophotometry and flame atomic absorption spectrometry difficult. Although an automated spectrophotometric flow injection procedure based on the thiocyanate method with solvent extraction was developed in this * Corresponding author.
S S D I 0039-9140(95)01688-0
laboratory [3], alternative methods exploiting kinetic catalysis have also been investigated [4-6]. The spectrophotometric method based on the catalytic effect of Mo(VI) ions in the oxidation of iodide by hydrogen peroxide [4] has been suggested to be very promising for the determination of molybdenum in biological materials [5-7]. The produced triiodide ion is spectrophotometrically monitored under the same conditions for the catalytic (presence of molybdenum) and non-catalytic (blank) reactions, and the difference between the absorbance signals is proportional to the molybdenum content in the sample. However, selectivity appears to be the main limitation, as other ions have similar catalytic properties. Svehla and Erdey [8] pointed out that interferences of several chemical species in the iodide-
2022
E.N.V.M. Carrilho et aL / Talanta 42 (1995) 2021-2026
hydrogen peroxide reaction were mainly due to the different reaction rates for triiodide formation in the presence and absence of molybdenum. This indicator reaction has been used in continuous flow systems for molybdenum determination in plant digest by different procedures, but prior iron separation by precipitation as hydroxide [5], on-line iron separation with ion exchange resin [6], or solvent extraction [9] have been needed. Masking agents for iron, such as fluoride in air-segmented flow systems [10], and sulphosalicylic acid in flow injection amperometry [11], have also been recommended. The aim o f the present paper is to describe an alternative procedure for the spectrophotometric catalytic determination of molybdenum by using a flow injection system that does not require previous iron separation or on-line molybdenum concentration. Amongst the several reagents evaluated in the present work, sulphosalicylic acid proved to be suitable for the determination o f molybdenum in the presence o f iron.
2. E x p e r i m e n t a l
2.1. Apparatus
The equipment consisted o f a model 432 Femto spectrophotometer (S3.o Paulo, Brazil) furnished with a tubular flow cell (12 mm optical path, 70 lal inner volume) from the same manufacturer and coupled to a REC 61 Radiometer recorder, an Ismatec m p l 3 R peristaltic pump with Tygon tubes, and other accessories. A manual sliding bar injector with two commutation sections [12], 0.5 mm i.d. Tygon microbore tubing (Norton Performance Plastics), and y-connections made from perspex were used to build the manifold.
Mo m l - i ) were prepared in 0.1% v/v nitric acid by appropriate dilutions of the stock solution. For selectivity evaluation, solutions with 5 . 0 - 5 0 . 0 m g F e l -~ (Fe203), 1.0mg V I -I (NH4VO3), 10.0 mg Cr 1-1 ( K 2 C r O 4 ) ' 500 mg Mg 1-1 (MgSO4 • 7H20), 500 mg AI 1- i (AICI3 • 6H20), 500 mg P 1-1 (Na2HPO4) or 1000 mg Ca 1-i (CaCO3) were also prepared in 0.1% v/v nitric acid. Potassium iodide solution 2.0% w/v (RI, Fig. 1) was prepared by dissolving 2.5g KI in 100 ml o f water and hydrogen peroxide solution 0.025% v/v (R2, Fig. 1) was prepared daily from a 30% H202 solution. The sample carder stream (C, Fig. 1) was a 0.1% v/v nitric acid solution. A 0.1 M sulphosalicyclic acid solution (M, Fig. 1) was prepared by dissolving 2.54g c7n606S • 2 H 2 0 in 100 ml of water. Biological samples were dry ashed according to the following procedure. 2.000 g o f ground (60 mesh) and oven-dried (60"C to constant weight) samples was ashed in a porcelain crucible for 4 h at 5500C. The ash was moistened with a few drops of water and l0 ml o f 4 M nitric acid was added; the mixture was then dried at 60"C. The residue was taken up with successive portions of 10-15 ml o f 0.1 M nitric acid solution, filtered through Whatman filter paper into a 100 ml volumetric flask, and made up to volume with the same acid. Before using, the solution was left to stand for about 12h.
M W~ C~
R2 H~O
2.2. Reagents, standards and samples
All chemicals were o f analytical-grade quality and freshly distilled-deionized water was used throughout. A 1000mgl -I molybdenum stock solution was prepared by dissolving 0.9201g of (NH4)6Mo7024" 4 H 2 0 in 500ml of 1% v/v HCI. Working standard solutions (0.0-100.0ng
Fig. 1. Diagram of the flow set-up for molybdenum (and iron) determination. B], B2 and B~ffi 10, 20 and 250cm reaction coils; D=speetrophotometer at 350nm for molybdenum and/or 490 nm for iron; S ffisample, aspi. rated at 2.6 ml min- ~; L ffisampling loop (200p.I); R~ =0.025% v/v hydrogen peroxide solution at 0.4 ml min-~; R2 ffi2.5% w/v potassium iodide solution at 0.8 ml min-~, M ffi0.1 M sulphosalicylic acid flowing at 0.3 ml min- ~; W = waste; x, y and z = confluence points; Cfsample carrier stream (0.1% v/v HNO.O at 1.6 ml rain- ~; H20 ffi water at 2.6 ml rain- ~.
E.N.V.M. Carrilho et al. / Talanta 42 (1995) 2021-2026
2.3. The flow injection system With the system in the situation depicted in Fig. 1, the sample is continuously aspirated to fill the sampling loop, and the sample carrier stream and reagents are continuously flowing towards detection. Water flows to improve system washing by dissolving any triiodide adsorbed in the inner walls of the analytical path. After introducing the selected sample volume into the sample carrier stream, the water stream recycles in its reservoir. The masking agent is added via a confluence point located in the commutator at a suitable flow rate to avoid excessive dispersion-dilution of the sample zone in the Bj reactor where the masking reaction takes place. The same criterion was used for predefining R~ and R2 flow rates and to provide suitable mixing conditions inside the B2 and B3 helical coils. Thereafter, the sample zone passes through the detector where the absorbance at 350 nm is measured for molybdenum (plus unmasked iron) and at 490 nm for the iron-salicylate complex. The system was dimensioned to provide the best figures of merit for molybdenum. For iron determination at 490 nm, further system optimization was not carried out because the sensitivity was already suitable.
2.4. Figures of merit The detection limits for molybdenum and iron were determined after 20 consecutive measurements of the blank solution (0.1% v/v nitric acid) according to IUPAC [13]. The accuracy for molybdenum determination was assessed by analyzing the same digestes by graphite furnace atomic absorption spectrometry in a Varian SpectrAA40 coupled with a GTA-96 furnace with pyrolytically coated graphite tubes and controlled by a DS-15 Date Station from the same manufacturer. The heating programme was adjusted accordingly. Accuracy assessment for iron determination was made with flame atomic absorption spectrometry with an air-acetylene flame.
3. Results and discussion
Concentrations of hydrogen peroxide (0.025% v/v) and potassium iodide (2.5% w/v) and the final pH of the reaction medium were selected by taking into account the ratio be-
2023
tween catalytic and non-catalytic signlas and the detection limit. Although the catalytic reaction rate increased with the concentrations of either hydrogen peroxide or potassium iodide, the non-catalytic signal (baseline) also increased. Similar behavior was observed earlier [5]. The pH of the reaction mainly affects the non-catalyzed reaction, i.e. it does not limit sensitivity for reaction pH values lower than two, and the lower the pH value the lower the baseline signal. Therefore, during sample pretreatment it was decided that the final acidity of the digest should be 0.1% v/v nitric acid. To avoid pH gradients along the sample zone, the same acid solution was used as carrier stream. In doing this, the pH of the solution leaving the flow cell was about 1.6. Preliminary experiments were carried out with several masking agents for iron. Tartrate, citrate, triethanolamine, pyrophosphate and oxalic acid minimized or even suppressed the interference of up to 15 mg Fe 1- t in the determination of 100lag M o l -~, but they also affected the catalytic reaction. Similar behaviour was observed with a 0.05% w/v sodium fluoride solution which caused a 90% decrease in iron interference and also affected the catalytic reaction rate leading to a 40% lowering in sensitivity. Higher fluoride concentrations did not improve the masking efficiency significantly, but decreased the reaction rate more than proportionally. Sulphosalicylic acid provided 85% masking efficiency, but with two advantages when compoared with fluoride and other iron-forming complexes. It did not change the catalytic reaction rate due to molybdenum, and iron could be determined as the iron(III)-sulphosalicylate complex [14] at 490 nm by using the same flow injection manifold. The system could also be used for the simultaneous determination of iron and molybdenum with a diode array detector, or sequentially with two spectrophotometers. The slope and linearity of the analytical calibration curves were not affected by the presence of up to 25mg1-1 iron when sulphosalicylic acid was used (Fig. 2). As a consequence, it was possible to infer that molybdenum could be determined in the presence of iron, because absorbances were additive. By knowing the iron content in the original sample, the absorbance due to molybdenum measured at 350 nm was corrected according to the following equations:
E.N.V.M. Carrilho et al. / Talanta 42 (1995) 2021-2026
2024
°f
0.8
8
0.6
0 I0.
I
0
10
I 20
I 30
I 40
J 50
molybdenum concentration, ng ml "~
Fig. 2. Molybdenum calibration curves with and without iron in the presence of 0.1 M sulphosalicylic acid (total absorbance values recorded at 350 nm): (am molybdenum alone (r = 0.9998); (b) molybdenum + 5 mg Fe 1-1 (r = 0.9997); (c) molybdenum + 25 mg Fe I - i (r = 0.9998). AMo = A M o + F e -
aFeCFe
C A M o --- AMo/amo
where AMo=absorbance at 350nm due to molybdenum, AMo+ ~:== absorbance at 350 nm due to molybdenum plus iron, aF= and aMo = iron and molybdenum absorptivities at 350 nm, CF= = iron concentration determined at 490 nm, and CMo = corrected molybdenum concentration. The influence of other elements on molybdenum determination (based on a mass/volume ratio of 1 g per 50 ml of the biological digest) at the 50 lag Mo 1-~ level was also investigated in the presence of sulphosalicylic acid at 350 and 490rim. as much as 1000mg Cal -m, 500mg Mgl -m, 500mg AII-m and 500mg P l-~ did not interfere with iron determination at 490 nm. As expected, interferences were observed for high concentrations of chromium (5mgl -m) and vanadium (0.5 mgl -m) only at
350 nm with signal enhancements not exceeding 20%. The influences of chromium(Vim and vanadium(V) are well-known since both elements catalyze the iodide oxidation by hydrogen peroxide [15]. Hopefully, chromium and vanadium do not occur at these levels in the digests, taking into account their maximum reported concentrations in dry plant tissues and food materials: i.e. 5 lag Cr g-1 and 10 lag Vg -~ [16-18]. The expected interference of phosphorus due to molybdophosphoric acid formation, also observed in earlier work [6], was only noticed for concentrations of phosphorus 10 times higher than its normal level in the digests, a 15% suppression in the molybdenum absorbance signal being observed in the presence of 250 mg P 1- =. Regarding tungsten(Vim and titanium(Vim, which also catalyze the indicator reaction, parallel experiments revealed that the system could tolerate their presence up to the maximum repored levels in biological materials [16]. Tungsten and titanium did not interfere at concentrations lower than 0.2mgl -= and 1 mg 1-~ respectively. These interferences are also discussed elsewhere [9,19]. There is good agreement between molybdenum concentration values obtained with the proposed flow injection catalytic method and graphite furnace atomic absorption spectrometry (Table I). In addition, Table 2 shows recoveries of about 100% for the same sample digests referred to in Table 1, which is also a good indication of the accuracy of the proposed method. The relative standard deviation of measurements (n = 5) for digests containing molybdenum within the range 10- 50 ng ml- m did not exceed 2%. The detection limit was 0.6 ng Mo ml -I for a 200 lai sample volume, and a throughput of 100 determinations per hour (50 per hour for molybdenum and 50 per hour for iron) was typically obtained. The flow system proved to be quite stable, simple and
Table 1 Comparison o f procedures for the determination of molybdenum in plant and food digests. Data are averages of three replicates Sample
m g M o k g - ~ (dry matter basis)
FIA Coffee leaves Soya flour Soybean leaves Rice leaves
0.249 0.761 0.368 0.282
mg Fe k g - ) FIA
GFAAS + + + +
0.001 0.007 0.003 0.002
0.287 0.809 0.402 0.297
+ 0.020 _+ 0.036 + 0.027 + 0.022
425 + 4.5 < 100 225 + 3.2 221 + 2.9
E.N.V.M. Carrilho et al. / Talanta 42 (1995) 2021-2026
2025
Table 2 Molybdenum recoveries from biological digests. Data are averages of three replicates Sample
Original concentration (ng Mo m l - i )
After addition (ng Mo m l - ~)
Recovery (%)
% r.s.d. (n = 5)
Coffee leaves
5.0 5.0 15.2 15.2 7.4 7.4 5.6 5.6
15.0 25.0 25.2 35.2 17.4 27.4 15.6 25.6
100.0 100.0 96.2 94.4 101.3 103.3 103.9 101.0
1.7 0.0 0.0 2.9 1.7 0.0 0.0 2.0
Soya flour Soybean leaves Rice leaves
Acknowledgments 04
o
I J
I0 rain
I q
490 nm
I FQ
OO Oh
b
~5Ortn
O.O
Fig. 3. Recorder tracing of a routine run. All measurements in triplicate. (a) Recording at 490 nm. From right to left, four iron standard solutions (5.0, 10.0, 15.0 and 2 0 . 0 m g l - t ) , and eight samples (the last two with iron contents lower than the detection limit). (b) Recording at 350 nm. From right to left, seven molybdenum standard solutions (0.0, 10.0, 20.0, 40.0, 60.0, 80.0 and 100.0 lag l - t ) eight samples, and five iron standard solutions (0.0, 5.0, 10.0, 15.0 and 20.0 mg 1-*).
reasonably robust, and can be used for most plant tissues, food and other biological products, where iron content in the digest does not exceed 25 mg 1-~ (1200 mg Fe g-~ dry matter). The calculated detection limit for iron was 0.2 mg 1- ~ at 490 nm. Samples with higher iron contents are not common and were not found for validation but limitations due to the linearity of the analytical calibration curves are expected. In this situation, the injected volume could be reduced accordingly for iron on-line dilution, with a proportional lack of molybdenum sensitivity. A typical recorder tracing relating to molybdenum and iron determination in plant digests is shown in Fig. 3.
The authors are grateful to F I N E P (Financiadora de Estudos e Projetos-PADCT II, processo 65.91.0324.00) for financial support, and to CNPq (Conselho Nacional de Desenvolvimento Cientifico e Technol6gico) for grants to E.N.V.M.C., F.J.K and E.A.G.Z. Thanks are also due to M.M. Silva for helping with the G F A A S measurements. At the time of this work, E.N.V,M.C. was a graduate student at the Institute de Quimica de S~o Carlos, Universidade de S~o Paulo, S~o Carlos-SP, Brazil.
References [1] D.R. Hoagland, Soil Sei., 60 (1945) 119. [2] V. Sauchelli, Trace Elements in Agriculture, Van Nostrand, New York, 1969. [3] H. Bergamin Filho, J.X. Medeiros, B.F. Reis and E.A.G. Zagatto, Anal. Chim. Acta, 101 (1978) 9. [4] K.B. Yatsimirskii and L.P. Afana's Eva, Zh. Anal. Khim., 11 (1956) 319. [5] F. Zhao-Lun and X. Shu-Kun, Anal. Chim. Acta, 145 (1983) 143. [6] L.C.R. Pessenda, A.O. Jacintho and E.A.G. Zhatto, Anal. Chim. Acta, 214 (1988) 239. [71 J.C. Andrade, S.P. Eiras and R.E. Bruns, Anal. Chim. Acta, 225 (1991) 149. [8] G. Svehla and L.Erdey, Microchim. J., 7 (1963) 206. [9] J.C. Andrade, R.E. Bruns and S.P. Eiras, Analyst, 118 (1993) 213. [10] M,H. Mahr and E. Pungor, Kem.-Kemi, 10 (1980) 585. [11] M, Trojanowicz, A. Hulanicki, W. Matuszewski, M. Palys, A. Fuksiewicz, T. Hulanicka-Michalak, S. Raszewski, J. SzyUer and W. Augustyniak, Anal. Chim. Acta. 188 (1986) 165. [12] F.J. Krug, H. Bergamin Fo and E.A.G. Zagatto, Anal. Chim. Acta, 179 (1986) 103. [13] Commission on Spectrochemical and other Optical Procedures for Analysis. Nomenclature, symbols, units and their usage in spectrochemical analysis-- I I, Spectrochim. Acta, 33 (1978) 241.
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[14] Z. Marczenko, Separation and Spectrophotometric Determination of Elements, Ellis Horwood, Chichester, 1986. [15] F. Eivazi, J.L. Sims and J. Crutchfield, Comm. Soil Sci., Plant Anal., 13 (1982) 135. [16] H.J.M. Bowen, Trace Elements in Biochemistry, Academic Press, London, 1966, pp. 61-84.
[17] J. Laporte, G. Kovacsik and J. Bellanger, Vegetable Matter, in M. Pinta (Ed.), Atomic Absorption Spectrometry, Adam Hilger, London, 1975, pp. 240-275. [18] Committee on Biological Effects of Atmospheric Pollutants, Chromium, National Academy of Sciences, Washington D.C., 1974. [19] B.F. Quin and P.H. Woods, Analyst, 104 (1979) 552.
Talanta 42 (1995) 2021-2026
Correction of iron interface in the spectrophotometric flow injection catalytic determination of molybdenum in plants Elma Neide Vasconcelos Martins Carrilho, Francisco Jos6 Krug *, Elias Ayres Guidetti Zagatto Centro de Energia Nuclear na Agricultura, Universidade de S~o Paulo, P.O. Box 96, 13400-970 Piracicaba, Sao Paulo, Brazil
Received 12 April 1995; revised 5 July 1995; accepted 7 July 1995
Abstract
Iron interference in the spectrophotometric catalytic determination of molybdenum based on the iodidehydrogen peroxide reaction can be corrected by using sulphosalicylic acid as masking and color-forming reagent. The catalytic influence of iron ions is circumvented to the extent of about 90% and correction of any remaining iron ions is possible by monitoring the colored iron(III)-salicylate complex at 490 rim. In this way, iron is also determined. With the proposed system, molybdenum can be determined in plant and food digests within the 0-100 I~g Mo 1-t range in the presence of up to 25 mg Fe !-t, at a sampling rate of about 50 determinations h-t. The relative standard deviation of 10 consecutive measurements was estimated as < 2%. Results for samples were comparable with those obtained by graphite furnace atomic absorption spectrometry. In addition, recoveries within the range 94-100% were calculated. Keywords: Iron interference; Molybdenum determination; Sulphosalicylic acid; Flow injection; Plants
1. Introduction
Molybdenum is an essential nutrient for animals and plants and its determination in plant, soil and food materials is o f utmost importance. Nevertheless, only a few well-equipped laboratories accomplish molybdenum determination on a routine basis. In general, molybdenum occurs in low concentrations in plant [1,2] ( 0 . 5 - 3 . 0 m g k g -I) and food materials, which makes the direct determination in the sample digest by simple techniques such as classical spectrophotometry and flame atomic absorption spectrometry difficult. Although an automated spectrophotometric flow injection procedure based on the thiocyanate method with solvent extraction was developed in this * Corresponding author.
S S D I 0039-9140(95)01688-0
laboratory [3], alternative methods exploiting kinetic catalysis have also been investigated [4-6]. The spectrophotometric method based on the catalytic effect of Mo(VI) ions in the oxidation of iodide by hydrogen peroxide [4] has been suggested to be very promising for the determination of molybdenum in biological materials [5-7]. The produced triiodide ion is spectrophotometrically monitored under the same conditions for the catalytic (presence of molybdenum) and non-catalytic (blank) reactions, and the difference between the absorbance signals is proportional to the molybdenum content in the sample. However, selectivity appears to be the main limitation, as other ions have similar catalytic properties. Svehla and Erdey [8] pointed out that interferences of several chemical species in the iodide-
2022
E.N.V.M. Carrilho et aL / Talanta 42 (1995) 2021-2026
hydrogen peroxide reaction were mainly due to the different reaction rates for triiodide formation in the presence and absence of molybdenum. This indicator reaction has been used in continuous flow systems for molybdenum determination in plant digest by different procedures, but prior iron separation by precipitation as hydroxide [5], on-line iron separation with ion exchange resin [6], or solvent extraction [9] have been needed. Masking agents for iron, such as fluoride in air-segmented flow systems [10], and sulphosalicylic acid in flow injection amperometry [11], have also been recommended. The aim o f the present paper is to describe an alternative procedure for the spectrophotometric catalytic determination of molybdenum by using a flow injection system that does not require previous iron separation or on-line molybdenum concentration. Amongst the several reagents evaluated in the present work, sulphosalicylic acid proved to be suitable for the determination o f molybdenum in the presence o f iron.
2. E x p e r i m e n t a l
2.1. Apparatus
The equipment consisted o f a model 432 Femto spectrophotometer (S3.o Paulo, Brazil) furnished with a tubular flow cell (12 mm optical path, 70 lal inner volume) from the same manufacturer and coupled to a REC 61 Radiometer recorder, an Ismatec m p l 3 R peristaltic pump with Tygon tubes, and other accessories. A manual sliding bar injector with two commutation sections [12], 0.5 mm i.d. Tygon microbore tubing (Norton Performance Plastics), and y-connections made from perspex were used to build the manifold.
Mo m l - i ) were prepared in 0.1% v/v nitric acid by appropriate dilutions of the stock solution. For selectivity evaluation, solutions with 5 . 0 - 5 0 . 0 m g F e l -~ (Fe203), 1.0mg V I -I (NH4VO3), 10.0 mg Cr 1-1 ( K 2 C r O 4 ) ' 500 mg Mg 1-1 (MgSO4 • 7H20), 500 mg AI 1- i (AICI3 • 6H20), 500 mg P 1-1 (Na2HPO4) or 1000 mg Ca 1-i (CaCO3) were also prepared in 0.1% v/v nitric acid. Potassium iodide solution 2.0% w/v (RI, Fig. 1) was prepared by dissolving 2.5g KI in 100 ml o f water and hydrogen peroxide solution 0.025% v/v (R2, Fig. 1) was prepared daily from a 30% H202 solution. The sample carder stream (C, Fig. 1) was a 0.1% v/v nitric acid solution. A 0.1 M sulphosalicyclic acid solution (M, Fig. 1) was prepared by dissolving 2.54g c7n606S • 2 H 2 0 in 100 ml of water. Biological samples were dry ashed according to the following procedure. 2.000 g o f ground (60 mesh) and oven-dried (60"C to constant weight) samples was ashed in a porcelain crucible for 4 h at 5500C. The ash was moistened with a few drops of water and l0 ml o f 4 M nitric acid was added; the mixture was then dried at 60"C. The residue was taken up with successive portions of 10-15 ml o f 0.1 M nitric acid solution, filtered through Whatman filter paper into a 100 ml volumetric flask, and made up to volume with the same acid. Before using, the solution was left to stand for about 12h.
M W~ C~
R2 H~O
2.2. Reagents, standards and samples
All chemicals were o f analytical-grade quality and freshly distilled-deionized water was used throughout. A 1000mgl -I molybdenum stock solution was prepared by dissolving 0.9201g of (NH4)6Mo7024" 4 H 2 0 in 500ml of 1% v/v HCI. Working standard solutions (0.0-100.0ng
Fig. 1. Diagram of the flow set-up for molybdenum (and iron) determination. B], B2 and B~ffi 10, 20 and 250cm reaction coils; D=speetrophotometer at 350nm for molybdenum and/or 490 nm for iron; S ffisample, aspi. rated at 2.6 ml min- ~; L ffisampling loop (200p.I); R~ =0.025% v/v hydrogen peroxide solution at 0.4 ml min-~; R2 ffi2.5% w/v potassium iodide solution at 0.8 ml min-~, M ffi0.1 M sulphosalicylic acid flowing at 0.3 ml min- ~; W = waste; x, y and z = confluence points; Cfsample carrier stream (0.1% v/v HNO.O at 1.6 ml rain- ~; H20 ffi water at 2.6 ml rain- ~.
E.N.V.M. Carrilho et al. / Talanta 42 (1995) 2021-2026
2.3. The flow injection system With the system in the situation depicted in Fig. 1, the sample is continuously aspirated to fill the sampling loop, and the sample carrier stream and reagents are continuously flowing towards detection. Water flows to improve system washing by dissolving any triiodide adsorbed in the inner walls of the analytical path. After introducing the selected sample volume into the sample carrier stream, the water stream recycles in its reservoir. The masking agent is added via a confluence point located in the commutator at a suitable flow rate to avoid excessive dispersion-dilution of the sample zone in the Bj reactor where the masking reaction takes place. The same criterion was used for predefining R~ and R2 flow rates and to provide suitable mixing conditions inside the B2 and B3 helical coils. Thereafter, the sample zone passes through the detector where the absorbance at 350 nm is measured for molybdenum (plus unmasked iron) and at 490 nm for the iron-salicylate complex. The system was dimensioned to provide the best figures of merit for molybdenum. For iron determination at 490 nm, further system optimization was not carried out because the sensitivity was already suitable.
2.4. Figures of merit The detection limits for molybdenum and iron were determined after 20 consecutive measurements of the blank solution (0.1% v/v nitric acid) according to IUPAC [13]. The accuracy for molybdenum determination was assessed by analyzing the same digestes by graphite furnace atomic absorption spectrometry in a Varian SpectrAA40 coupled with a GTA-96 furnace with pyrolytically coated graphite tubes and controlled by a DS-15 Date Station from the same manufacturer. The heating programme was adjusted accordingly. Accuracy assessment for iron determination was made with flame atomic absorption spectrometry with an air-acetylene flame.
3. Results and discussion
Concentrations of hydrogen peroxide (0.025% v/v) and potassium iodide (2.5% w/v) and the final pH of the reaction medium were selected by taking into account the ratio be-
2023
tween catalytic and non-catalytic signlas and the detection limit. Although the catalytic reaction rate increased with the concentrations of either hydrogen peroxide or potassium iodide, the non-catalytic signal (baseline) also increased. Similar behavior was observed earlier [5]. The pH of the reaction mainly affects the non-catalyzed reaction, i.e. it does not limit sensitivity for reaction pH values lower than two, and the lower the pH value the lower the baseline signal. Therefore, during sample pretreatment it was decided that the final acidity of the digest should be 0.1% v/v nitric acid. To avoid pH gradients along the sample zone, the same acid solution was used as carrier stream. In doing this, the pH of the solution leaving the flow cell was about 1.6. Preliminary experiments were carried out with several masking agents for iron. Tartrate, citrate, triethanolamine, pyrophosphate and oxalic acid minimized or even suppressed the interference of up to 15 mg Fe 1- t in the determination of 100lag M o l -~, but they also affected the catalytic reaction. Similar behaviour was observed with a 0.05% w/v sodium fluoride solution which caused a 90% decrease in iron interference and also affected the catalytic reaction rate leading to a 40% lowering in sensitivity. Higher fluoride concentrations did not improve the masking efficiency significantly, but decreased the reaction rate more than proportionally. Sulphosalicylic acid provided 85% masking efficiency, but with two advantages when compoared with fluoride and other iron-forming complexes. It did not change the catalytic reaction rate due to molybdenum, and iron could be determined as the iron(III)-sulphosalicylate complex [14] at 490 nm by using the same flow injection manifold. The system could also be used for the simultaneous determination of iron and molybdenum with a diode array detector, or sequentially with two spectrophotometers. The slope and linearity of the analytical calibration curves were not affected by the presence of up to 25mg1-1 iron when sulphosalicylic acid was used (Fig. 2). As a consequence, it was possible to infer that molybdenum could be determined in the presence of iron, because absorbances were additive. By knowing the iron content in the original sample, the absorbance due to molybdenum measured at 350 nm was corrected according to the following equations:
E.N.V.M. Carrilho et al. / Talanta 42 (1995) 2021-2026
2024
°f
0.8
8
0.6
0 I0.
I
0
10
I 20
I 30
I 40
J 50
molybdenum concentration, ng ml "~
Fig. 2. Molybdenum calibration curves with and without iron in the presence of 0.1 M sulphosalicylic acid (total absorbance values recorded at 350 nm): (am molybdenum alone (r = 0.9998); (b) molybdenum + 5 mg Fe 1-1 (r = 0.9997); (c) molybdenum + 25 mg Fe I - i (r = 0.9998). AMo = A M o + F e -
aFeCFe
C A M o --- AMo/amo
where AMo=absorbance at 350nm due to molybdenum, AMo+ ~:== absorbance at 350 nm due to molybdenum plus iron, aF= and aMo = iron and molybdenum absorptivities at 350 nm, CF= = iron concentration determined at 490 nm, and CMo = corrected molybdenum concentration. The influence of other elements on molybdenum determination (based on a mass/volume ratio of 1 g per 50 ml of the biological digest) at the 50 lag Mo 1-~ level was also investigated in the presence of sulphosalicylic acid at 350 and 490rim. as much as 1000mg Cal -m, 500mg Mgl -m, 500mg AII-m and 500mg P l-~ did not interfere with iron determination at 490 nm. As expected, interferences were observed for high concentrations of chromium (5mgl -m) and vanadium (0.5 mgl -m) only at
350 nm with signal enhancements not exceeding 20%. The influences of chromium(Vim and vanadium(V) are well-known since both elements catalyze the iodide oxidation by hydrogen peroxide [15]. Hopefully, chromium and vanadium do not occur at these levels in the digests, taking into account their maximum reported concentrations in dry plant tissues and food materials: i.e. 5 lag Cr g-1 and 10 lag Vg -~ [16-18]. The expected interference of phosphorus due to molybdophosphoric acid formation, also observed in earlier work [6], was only noticed for concentrations of phosphorus 10 times higher than its normal level in the digests, a 15% suppression in the molybdenum absorbance signal being observed in the presence of 250 mg P 1- =. Regarding tungsten(Vim and titanium(Vim, which also catalyze the indicator reaction, parallel experiments revealed that the system could tolerate their presence up to the maximum repored levels in biological materials [16]. Tungsten and titanium did not interfere at concentrations lower than 0.2mgl -= and 1 mg 1-~ respectively. These interferences are also discussed elsewhere [9,19]. There is good agreement between molybdenum concentration values obtained with the proposed flow injection catalytic method and graphite furnace atomic absorption spectrometry (Table I). In addition, Table 2 shows recoveries of about 100% for the same sample digests referred to in Table 1, which is also a good indication of the accuracy of the proposed method. The relative standard deviation of measurements (n = 5) for digests containing molybdenum within the range 10- 50 ng ml- m did not exceed 2%. The detection limit was 0.6 ng Mo ml -I for a 200 lai sample volume, and a throughput of 100 determinations per hour (50 per hour for molybdenum and 50 per hour for iron) was typically obtained. The flow system proved to be quite stable, simple and
Table 1 Comparison o f procedures for the determination of molybdenum in plant and food digests. Data are averages of three replicates Sample
m g M o k g - ~ (dry matter basis)
FIA Coffee leaves Soya flour Soybean leaves Rice leaves
0.249 0.761 0.368 0.282
mg Fe k g - ) FIA
GFAAS + + + +
0.001 0.007 0.003 0.002
0.287 0.809 0.402 0.297
+ 0.020 _+ 0.036 + 0.027 + 0.022
425 + 4.5 < 100 225 + 3.2 221 + 2.9
E.N.V.M. Carrilho et al. / Talanta 42 (1995) 2021-2026
2025
Table 2 Molybdenum recoveries from biological digests. Data are averages of three replicates Sample
Original concentration (ng Mo m l - i )
After addition (ng Mo m l - ~)
Recovery (%)
% r.s.d. (n = 5)
Coffee leaves
5.0 5.0 15.2 15.2 7.4 7.4 5.6 5.6
15.0 25.0 25.2 35.2 17.4 27.4 15.6 25.6
100.0 100.0 96.2 94.4 101.3 103.3 103.9 101.0
1.7 0.0 0.0 2.9 1.7 0.0 0.0 2.0
Soya flour Soybean leaves Rice leaves
Acknowledgments 04
o
I J
I0 rain
I q
490 nm
I FQ
OO Oh
b
~5Ortn
O.O
Fig. 3. Recorder tracing of a routine run. All measurements in triplicate. (a) Recording at 490 nm. From right to left, four iron standard solutions (5.0, 10.0, 15.0 and 2 0 . 0 m g l - t ) , and eight samples (the last two with iron contents lower than the detection limit). (b) Recording at 350 nm. From right to left, seven molybdenum standard solutions (0.0, 10.0, 20.0, 40.0, 60.0, 80.0 and 100.0 lag l - t ) eight samples, and five iron standard solutions (0.0, 5.0, 10.0, 15.0 and 20.0 mg 1-*).
reasonably robust, and can be used for most plant tissues, food and other biological products, where iron content in the digest does not exceed 25 mg 1-~ (1200 mg Fe g-~ dry matter). The calculated detection limit for iron was 0.2 mg 1- ~ at 490 nm. Samples with higher iron contents are not common and were not found for validation but limitations due to the linearity of the analytical calibration curves are expected. In this situation, the injected volume could be reduced accordingly for iron on-line dilution, with a proportional lack of molybdenum sensitivity. A typical recorder tracing relating to molybdenum and iron determination in plant digests is shown in Fig. 3.
The authors are grateful to F I N E P (Financiadora de Estudos e Projetos-PADCT II, processo 65.91.0324.00) for financial support, and to CNPq (Conselho Nacional de Desenvolvimento Cientifico e Technol6gico) for grants to E.N.V.M.C., F.J.K and E.A.G.Z. Thanks are also due to M.M. Silva for helping with the G F A A S measurements. At the time of this work, E.N.V,M.C. was a graduate student at the Institute de Quimica de S~o Carlos, Universidade de S~o Paulo, S~o Carlos-SP, Brazil.
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[14] Z. Marczenko, Separation and Spectrophotometric Determination of Elements, Ellis Horwood, Chichester, 1986. [15] F. Eivazi, J.L. Sims and J. Crutchfield, Comm. Soil Sci., Plant Anal., 13 (1982) 135. [16] H.J.M. Bowen, Trace Elements in Biochemistry, Academic Press, London, 1966, pp. 61-84.
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