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Determination of sulfur in food by high resolution continuum source flame molecular absorption spectrometry
Spectrochimica Acta Part B 101 (2014) 234–239
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Spectrochimica Acta Part B journal homepage: www.elsevier.com/locate/sab
Determination of sulfur in food by high resolution continuum source flame molecular absorption spectrometry☆ Elżbieta Zambrzycka, Beata Godlewska-Żyłkiewicz ⁎ Institute of Chemistry, University of Bialystok, Hurtowa 1, 15-399 Białystok, Poland
a r t i c l e
i n f o
Article history: Received 15 May 2014 Accepted 21 August 2014 Available online 10 September 2014 Keywords: Molecular absorption spectrometry Sulfur determination Food analysis
a b s t r a c t In the present work, a fast, simple and sensitive analytical method for determination of sulfur in food and beverages by high resolution continuum source flame molecular absorption spectrometry was developed. The determination was performed via molecular absorption of carbon monosulfide, CS. Different CS rotational lines (257.959 nm, 258.033 nm, 258.055 nm), number of pixels and types of standard solution of sulfur, namely: sulfuric acid, sodium sulfate, ammonium sulfate, sodium sulfite, sodium sulfide, DL-cysteine, and L-cystine, were studied in terms of sensitivity, repeatability of results as well as limit of detection and limit of quantification. The best results were obtained for measurements of absorption of the CS molecule at 258.055 nm at the wavelength range covering 3 pixels and DL-cysteine in 0.2 mol L−1 HNO3 solution as a calibration standard. Under optimized conditions the limit of detection and the limit of quantification achieved for sulfur were 10.9 mg L−1 and 36.4 mg L−1, respectively. The repeatability of the results expressed as relative standard deviation was typically b 5%. The accuracy of the method was tested by analysis of digested biological certified reference materials (soya bean flour, corn flour and herbs) and recovery experiment for beverage samples with added known amount of sulfur standard. The recovery of analyte from such samples was in the range of 93–105% with the repeatability in the range of 4.1–5.0%. The developed method was applied for the determination of sulfur in milk (194 ± 10 mg kg− 1), egg white (2188 ± 29 mg kg− 1), mineral water (31.0 ± 0.9 mg L− 1), white wine (260 ± 4 mg L− 1) and red wine (82 ± 2 mg L−1), as well as in sample rich in ions, such as bitter mineral water (6900 ± 100 mg L− 1). © 2014 Elsevier B.V. All rights reserved.
1. Introduction Sulfur, next to oxygen and silicon, is the most abundant constituent of minerals. Sulfur is also one of the major elements essential to life, as many functionally important cellular components, such as essential amino acids, contain this element [1]. Sulfur is mostly taken from food rich in proteins, such as eggs, fresh milk, red meat and fish [2]. There are also many vegetables containing high content of sulfur like sweet corn, spinach, onion and garlic and cruciferous vegetables (broccoli, cauliflower, cabbage, kale) (0.7–14 g kg−1) [3,4]. In mineral and drinking waters sulfur is mainly present as sulfates and sulfides, while in food of animal origin (chicken eggs, meat, milk) as sulfur-containing amino acids: methionine, cysteine, cystine and taurine. A variety of fruit, vegetables, and grains naturally include sulfur as organic compound — methylsulfonylmethane, also known as dimethyl sulfone [5]. Sulfur is often added to food as a preservative. Sulfur dioxide and sulfites, which
☆ Selected paper from the European Symposium on Atomic Spectrometry ESAS 2014 & 15th Czech–Slovak Spectroscopic Conference, Prague, Czech Republic, 16–21 March 2014. ⁎ Corresponding author. Tel.: +48 85 745782; fax: +48 85 7470113. E-mail address: [email protected] (B. Godlewska-Żyłkiewicz).
http://dx.doi.org/10.1016/j.sab.2014.08.041 0584-8547/© 2014 Elsevier B.V. All rights reserved.
release sulfur dioxide in acidic solution, are chemical additives used for inhibiting undesirable bacterial growth and oxidation processes in wine production [6,7]. The maximum permitted amount of sulfur in wine and drinking water in various countries is strictly regulated by legislation concerning food safety. The maximum contaminant level of sulfate in drinking water in Poland is 250 mg L−1, while the maximum limit of sulfides is only 0.05 mg L−1[8]. In the European Union the maximum limits for the total SO2 in red and white wines are 150 and 200 mg L−1 (dry wine), 200 and 250 mg L−1 (medium wine) and 300 and 400 mg L−1 (sweet wine), respectively [6]. The determination of sulfur is of interest in many areas such as human health, agriculture and food industries [9]. Among the classical methods of sulfur determination are gravimetric analysis based on precipitation of barium sulfate and combustion to sulfur dioxide with detection by titration, thermal conductivity, flow-injection turbidimetry [10], and ultraviolet fluorescence spectrometry [11]. Other methods used include wavelength dispersive X-ray fluorescence spectrometry [11,12], spectrophotometry [13], ion chromatography [14], inductively coupled plasma optical emission spectrometry [15–17] and inductively coupled plasma mass spectrometry [18,19]. Nevertheless, each of these methods has its own methodological limitations, such as poor precision and selectivity resulting from a complex chemistry of this element.
E. Zambrzycka, B. Godlewska-Żyłkiewicz / Spectrochimica Acta Part B 101 (2014) 234–239
The development of high resolution continuum source atomic absorption spectrometry (HR CS AAS) introduced new possibilities in the determination of sulfur and other non-metals such as phosphorus and halogens (Cl, I, Br) based on absorption spectra of formed molecules [20–26]. The first application of the HR CS AAS instrument for the determination of sulfur in wine was done in flame by Huang et al. [27–29]. The studies performed using different continuum source spectrometers developed at the ISAS Berlin [28,29] have shown, that in the spectral range between 200 and 465 nm several molecular bands can be found. It was proved that the spectra band around 258 nm appears due to the presence of carbon monosulfide (CS) molecular absorption, whereas at around 325 nm it appears due to the absorption of the SH molecule. Since CS is a more stable molecule than SH (the bond enthalpies of C–S and S–H bonds are equal to 714.1 and 344.3 kJ mol−1), the molecular absorption of CS is mostly used for sulfur determination. During the last decade a number of papers reporting the determination of sulfur based on CS spectra using commercially available HR CS AAS spectrometers equipped with a flame [30–34] or graphite furnace [34–38] have been published. The volatility of various sulfur chemical forms is highly variable, hence careful optimization of flame conditions, chemical modifiers and furnace temperature as well as type of sulfur standard is essential for accurate results of analysis. Quantitative analysis was performed by standard addition [26,27,29,34] or external calibration graph technique [31,34,35,37]. Different inorganic compounds containing sulfur were tested as standards, such as H2SO4[32], Na2SO4[32], MgSO4·7H2O [30,33,38], (NH4)2SO4[31], Na2SO3[29], and Na2S [35]. Among them sulfates were preferred for determination of CS in flame, as they are stable under various conditions (acidity, content of oxidizing agents, ethanol, etc.) (see Table 1) [28]. Kowalewska [34] tested various organic compounds, dibuthyl sulfide ((C4H9)2S), tertnonyl polysulfide ((C6H13C(CH3)2)2Sx), carbon disulfide (CS2), as standards for the determination of sulfur in petroleum products by HR CS FMAS and HR CS GFMAS, but the accurate results were only obtained using heavy petroleum products with known sulfur content for calibration. Other organic sulfur compounds, such as thiourea [36], and L-cysteine [38] were used as standards during the determination of sulfur by slurry [39] and solid sampling [35,36,38] HR GF MAS. The thiourea standard in the presence of Pd [36] modifier was employed for sulfur determination in biological samples, while L -cysteine standard was used for its determination in coal [38]. It was found that Lcysteine behavior during the pyrolysis process was similar to the behavior of the coal sample [38]. All of these methods were used for the analysis of samples containing sulfur at the levels of 0.03–6%. The aim of this work was to develop a simple method suitable for the determination of sulfur in food/beverage samples by HR CS FMAS after minimal sample pre-treatment (dilution with appropriate solvent).
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The type of standard solution suitable for such analysis was selected. The influence of matrix constituents on the analytical signal of different standards was investigated. During optimization of experimental parameters the best conditions in terms of sensitivity, repeatability of results, limit of detection and limit of quantification, as well as accuracy of results were always selected. 2. Experimental 2.1. Instrumentation and flame conditions All the absorption measurements were performed using a high resolution continuum source atomic absorption spectrometer ContrAA® 300 (Analytik Jena AG, Jena, Germany) equipped with a continuum light source — xenon short-arc lamp XBO 301 (GLE, Berlin, Germany) with the arc in a hot spot mode suitable for all elements determination, a double monochromator consisting of a prism pre-monochromator and a high resolution echelle grating monochromator and a charge-coupled device (CCD) array detector with 588 pixels. The detector allows for the visualization of the environment of the analytical line using 200 pixels. An air-acetylene flame was used for the determination of sulfur via CS molecule under optimized conditions (burner height: 8 mm, burner length: 100 mm, air–C2H2 flow rate: 120 L h−1). The digestion of samples was performed in a microwave system 150 Ethos Plus (Milestone, Italy). 2.2. Reagents and analytical solutions All reagents used were of analytical grade or higher. Sulfur standard solutions were prepared by dissolution of sulfuric acid (95%, POCH, Poland), high purity ammonium sulfate anhydrous (POCH, Poland), sodium sulfate anhydrous (Sigma-Aldrich, USA), sodium sulfite anhydrous (POCH, Poland), sodium sulfide non-hydrate (Sigma-Aldrich, USA) in Milli-Q water obtained using a Milli-Q system (Millipore, USA). L-Cystine (Sigma-Aldrich, USA) and DL-cysteine (Sigma-Aldrich, USA) were dissolved in 0.2 mol L−1 HNO3 (69.5%, Trace Select, Fluka, France). Hydrochloric acid (37%, fuming, Trace Select, Fluka, France) and sodium chloride (anhydrous, Sigma-Aldrich, USA) were used for milk and chicken egg white preparation. Nitric acid (69.5%, Trace Select, Fluka, France) and hydrogen peroxide (30%, Merck, Germany) were used for the digestion of plant samples. Sodium hydrogen carbonate (POCH, Poland), ethanol (99.8%, POCh, Poland), lead(II), calcium and magnesium nitrates (POCH, Poland) were used to study the matrix interference. The chicken eggs, pasteurized cow's milk with 0.5% milk fat, Hungarian bitter water “Hunyadi Janos”, mineral water “Saguaro”, Hungarian semi-dry white wine “Tokaj Furmint”, Bulgarian semi-dry red wine “Sophia Sakar”, samples of which were investigated in this
Table 1 Literature data on sulfur determination based on molecular absorption of CS using HR CS FMAS. Wavelength Number of pixels
Calibration standard
258.055 nm
3
DL-cysteine, L-cystine,
H2SO4, Na2SO4, (NH4)2SO4, Na2SO3, Na2S
258.056 nm 257.593 nm 258.055 nm
3 5
(NH4)2SO4, H2SO4, KHSO4, Na2SO3, (NH4)2S, Na2S2O3 MgSO4·7H2O
257.595 257.958 258.056 258.056
nm nm nm nm
1
(NH4)2SO4
5
257.595 nm
5
H2SO4,Na2SO4, (NH4)2SO4, Na2S2O8 MgSO4·7H2O
258.056 nm 11
Petroleum products, (C4H9)2S, CS2, (C6H13C(CH3)2)2Sx
Limit of detection
RSD
Samples
10.9 mg L−1 b5.8% Chicken egg white; fresh milk; wine; bitter and mineral water; CRM: soya bean flour (INCT-SBF-4); mixed polish herbs (INCT-MPH-2); corn flour (INCT-CF-3); tomato leaves (NIST-1573a) 2.4 mg L−1 – CRM: cast iron (EURONOM ZRM 428-2); cast iron 4.4 mg L−1 – (EURONOM ZRM 484-1) 22.4 mg L−1 b5.7% Orange leaves; CRM: tomato leaves (NIST 1573a); peach leaves (NIST 1547); spinach (NIST 1570a) 15.1 mg L−1 4.8% Fungicide, fertilizer −1 22.4 mg L 5.7% 21.8 mg L−1 4.2% 100 mg L−1 b9.7% Coal, CRM: coal (NIST 1632b) 34.7 mg L−1 – 21 mg L
−1
–
In bold — calibration standard used for quantitative analysis; RSD - relative standard deviation.
Ref. This paper [28] [30] [31]
[32]
Medicinal plants; CRM: spinach leaves (NIST 1570a); apple leaves [33] (NIST 1515) Crude oil; petroleum products; CRM: residual fuel oil (1634b, 1618, JPI); [34] fuel oil (F007, F009)
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study, were locally available brands, collected in supermarkets in Białystok (Poland). The accuracy of the HR CS FMAS method was checked by analyzing certified reference materials (CRM) of soya bean flour (INCT-SBF-4), mixed polish herbs (INCT-MPH-2), corn flour (INCT-CF-3) supplied by the Institute of Nuclear Chemistry and Technology (Warsaw, Poland) and tomato leaves (NIST-SRM-1573a) supplied by the National Institute of Standards and Technology (Gaithersburg, Maryland, USA).
2.3. Analytical procedure The rotational line at 258.055 nm was chosen for measurements of absorption of the CS molecule. For the evaluation of the absorption signals, the integrated absorbance of three pixels (central pixel ± 1), which covers a range of approximately 6 pm, was used. All absorbance values are mean values based on three repetitive measurements. The dynamic background correction technique with reference was used. The quantitative determination of sulfur was carried out by the external calibration graph technique based on DL-cysteine standard. During the optimization of each parameter the best conditions in terms of sensitivity, repeatability of results as well as limit of detection were always selected. Limits of detection (LOD) and quantification (LOQ) were calculated according to following equations: LOD = 3SDblank / a, LOQ = 10SDblank / a, where “SDblank” is standard deviation of the absorbance measurements of blank solution (0.2 mol L− 1 HNO3) and “a” is a slope of the calibration graph.
2.4. Preparation of samples The egg white was prepared by a modified procedure developed by Awadé and Efstathiou [40]. The egg white was manually separated from the egg yolk, introduced to plastic vessel (90 mL) and weighed. Next, the egg white was diluted 1:1 with an equivalent amount of 0.1 mol L−1 NaCl and homogenized with a magnetic stirrer for 3 h. For the absorbance measurements 1 g (~ 1 mL) aliquots of obtained solutions were transferred to smaller plastic vessels (20 mL) and diluted with 0.1 mol L−1 NaCl to 5 mL, 10 mL or 20 mL. Prior to analysis the 1.0 g samples of pasteurized cow's milk were diluted to 3 mL with 0.1 mol L− 1 HCl and then homogenized with a magnetic stirrer for 5 min. The samples of waters and wine were acidified with HNO3 (to obtain final acid concentration of 0.2 mol L− 1) and next degassed under a vacuum for 1 h. All certified reference materials were digested before analysis in a closed microwave system. About 0.5 g each of the CRM plant materials was placed into a Teflon vessel and then 6 mL of concentrated HNO3 and 1 mL of H2O2 were added. The heating program included the following steps (power/temperature/hold time): step 1: 250 W/ 110 °C/2 min; step 2: 0 W/110 °C/2 min; step 3: 250 W/185 °C/6 min; step 4: 400 W/185 °C/5 min; and step 5: 600 W/185 °C/5 min. The obtained solutions were transferred to quartz crucibles, evaporated near to dryness and next diluted with 0.2 mol L−1 HNO3 to 10 mL.
3.1. Optimization of signal evaluation mode The absorption spectrum of the CS molecule produced in an air– acetylene flame registered around 258 nm under optimized burner parameters is presented in Fig. 1. This band shows several sharp rotational lines, among which the strongest lines were observed at 257.959 nm, 258.033 nm and 258.055 nm. Different rotational lines were used for the determination of sulfur by other authors [21,28,30–35] because they vary in the sensitivity and the possible risk of occurrence of interferences from e.g. Fe [26,28] or Co [23]. Additionally, by taking more CS lines into account, i.e., summing up the integrated absorbance of individual rotational lines, the sensitivity as well as the signal-to-noise ratio could be significantly improved as was reported in [21,26,33]. In this work the calibration graphs for (NH4)2SO4 standard were constructed on the basis of absorbance signals registered for each individual line at CP (central pixel) and for the sum of absorbances of three most sensitive lines (at 257.959, 258.033 and 258.055 nm). The data are shown in Table 2. The sensitivity of measurements for individual lines was similar, but the lowest limit of detection was obtained for measurements at 258.055 nm. The sensitivity of measurements based on the sum of absorbances increased by a factor of 3, while the LOD value was from 2 to 5 times lower than these calculated for individual lines (Table 2). These results are in agreement with the results obtained by Heitmann et al. [26] and Bechlin et al. [33]. Such positive effect was observed for summing of the most sensitive lines. However, the summing of more than 3 absorption lines, which differ in shape, neighborhood and sensitivity, improved the sensitivity of measurements in a lesser extent. It was found that for the summing of 6 and 9 lines the sensitivity increased by factors of 5 and 6.8 in comparison to the measurements of one individual line, but the limit of detection was higher due to the higher noise level. For practical purposes only the single line at 258.055 nm was chosen for sulfur determination in further work. The sensitivity of measurements may be improved by increasing the number of pixels used for detection. The influence of wavelength integrated absorbance on the sensitivity and precision was studied by varying the number of pixels used for detection from 1 to 7. The slopes and correlation coefficients of the calibration graphs, limits of detection, limits of quantification and repeatability for different number of pixels are summarized in Table 2. It was observed that the sensitivity of measurements when 5 pixels were taken for signal evaluation was 2.9 times higher in comparison to that obtained for the evaluation of 1 pixel and 20% higher than for the evaluation of 3 pixels. However, the LOD value calculated using 5 pixels was worse than that obtained using 3 pixels. Using 7 pixels the sensitivity was similar to that obtained for 5 pixels,
3. Results and discussion The method for sulfur determination in food is based on the measurements of the absorption band of the stable diatomic CS molecule by HR CS FMAS. The formation of the CS molecule in a flame is promoted by using an excess of acetylene, which produces a reducing environment of the flame. The initial optimization experiments were performed using (NH4)2SO4 as sulfur standard. The obtained data were always analyzed in terms of sensitivity, repeatability (calculated as RSD), limit of detection (LOD) and limit of quantification (LOQ) values.
Fig. 1. Absorption spectrum of the CS molecule with the strongest rotational lines: 1) 258.055 nm, 2) 258.033 nm and 3) 257.959 nm, obtained for a DL-cysteine solution containing 600 mg L −1 of S.
E. Zambrzycka, B. Godlewska-Żyłkiewicz / Spectrochimica Acta Part B 101 (2014) 234–239 Table 2 Influence of the CS rotational line, summation of absorbance signals of rotational lines (Σ lines) and number of pixels on the sensitivity, linear correlation coefficient of calibration graph (R), limit of detection (LOD), limit of quantification (LOQ) obtained for (NH4)2SO4 as sulfur standard (n = 3). Wavelength
Number of pixels
Slope
R
LOD
LOQ −1
mg L 257.959 258.033 258.055 Σ lines 258.055
nm nm nm nm
1 1 1 1 3 5 7
1.4·10−5 1.4·10−5 1.5·10−5 4.3·10−5 3.6·10−5 4.4·10−5 4.3·10−5
0.9993 0.9998 0.9998 0.9998 0.9998 0.9989 0.9998
27.8 31.3 12.0 5.6 10.0 12.9 10.9
92.8 104.3 40.0 18.8 33.3 42.9 36.3
but the noise that is computed to the total integrated absorbance increased (relative standard deviation of measurements reached 17%). For further studies the absorbance values at 258.055 nm based on the detection of 3 pixels were used, due to good sensitivity, precision and the lowest LOD value. The limit of detection for sulfate standard (10.0 mg L− 1) under optimized measurement conditions is lower in comparison to the values reported in the literature (Table 1). 3.2. Standard solutions for calibration It was observed that the sensitivity of the CS molecular absorption produced in the air–acetylene flame depends on the chemical form of the sulfur-containing substances [28,34]. As was mentioned earlier different standards were tested for quantitative analysis of sulfur, but irrespective of the type of analyzed samples, mainly sulfates were chosen for calibration purposes (Table 1). The properly optimized analytical procedure should provide conditions for achieving proper stabilization of sulfur in flame regardless of the chemical form in which sulfur is present in the analyzed samples. This aspect was investigated in more detail in the current work. Initially the influence of different concentrations of nitric acid (in the range of 0.01–1 mol L− 1) on the CS molecular absorbance obtained using different sulfur containing standards, namely sulfuric acid, sodium sulfate, ammonium sulfate, sodium sulfite, sodium sulfide, DL-cysteine, and L-cystine, was studied. It must be noticed that due to low solubility of L-cystine in water it must be dissolved in acidic conditions. Addition of 0.04 mol L− 1 HNO3 to aqueous solution of L-cystine caused 40% increase of absorbance of sulfur. Further acidification of this standard solutions caused only a slight increase of absorbance. The absorbance of DL-cysteine and Na2SO4, after addition of 0.01 mol L−1 HNO3 increased by 20% in comparison with aqueous standard. The absorbance of H2SO4 and (NH4)2SO4 solutions remained constant in the studied range of HNO3 concentration. The effect of the rapid increase of absorbance for sulfite (1000%) and sulfide (400%) standards in the presence of nitric acid at a concentration above 0.04 mol L−1 was observed, which was also reported by Huang et al. [27,28]. In order to avoid the differences in the formation of CS molecule from standards and digested samples all solutions were further prepared in 0.2 mol L−1 HNO3. The parameters of the calibration graphs using different sulfur containing compounds, limits of detection, limits of quantification and repeatability for different standard concentrations (data presented in Supplementary Table S1 (Appendix A)) were analyzed. The highest sensitivity of the calibration graphs was obtained using solutions containing sulfur in the form of sulfite. Huang el al. [28] explained that effect as the generation of gaseous SO2 during sample nebulization and the more efficient transport into the flame, as compared with the ionic forms of sulfur. However, the sulfites present originally in samples might be converted into sulfates when the medium contains oxidizing compounds (e.g. during sample digestion step with HNO3 and H2O2) or into SO2 if it is very acidic or alkaline. Hence, this standard is not recommended for analysis of digested samples. Sodium sulfide in acidic solution is
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converted into volatile H2S, which is easily transferred into flame. It was observed that, during storage of such solutions gaseous H2S is continuously removed from the analyzed samples. After 15 min of storage the signal of sulfur decreased by 25%, while after 40 min it decreased by almost 80%. For these reasons, although the highest sensitivities and lowest limits of detection were obtained for Na2SO3 and Na2S solutions, these compounds are not suitable as standards for the determination of sulfur in real biological samples by HR CS FMAS (see Table 3). Baysal and Akman [32] have found the best sensitivity and wide linear range of the concentration graph (0.005–20% of S) using sulfuric acid as a standard. However, the increasing concentration of sulfuric acid in standards causes a rapid decreasing of solution pH, which may change the conditions of the formation of CS molecule. For this reason we excluded this standard from further investigations. Similar sensitivities as well as limits of detection were obtained for all other sulfur standards, but the best repeatability of results was observed for DL-cysteine and (NH4)2SO4 standards. The influence of the possible matrix constituents of analyzed samples on the CS molecular absorbance was examined and is shown in Fig. 2. The absorbance of organic standards of sulfur (DL-cysteine, L-cystine) remained practically constant (differences b 5%) in the presence of 10% −1 hydrogen peroxide, 10% ethanol, 70 mg L−1 HCO− 3 and 0.1 mol L NaCl. Greater effects were observed for (NH4)2SO4 standard solution (differences in the range of 5–15%). The high resolution of applied spectrometer significantly minimizes the possibility of spectral interferences from foreign metals, but does not eliminate the occurrence of nonspectral interferences. The lowest influence of Pb2+, Mg2+ and Ca2+ ions was observed on the signal of DL-cysteine. This suggests, that a biological matrix, containing high content of carbon, might be responsible for better formation and better thermal stability of the CS molecule. Kowalewska [34] has also observed positive effect of organic matrix (heavy petroleum products) on the efficiency of CS formation in flame. Similar effect was also found in graphite furnace by Ferreira et al. [36] during analysis of biological samples and Kowalewska [34] during analysis of heavy petroleum products. During the analysis of coal samples losses of sulfur from standard based on MgSO4, due to the formation of less stable competitive molecule in the presence of coal carbon, were reported, therefore L-cysteine standard was applied for such analysis [38]. Different standards were used for the determination of sulfur in CRMs of biological samples (Table 3). It was confirmed that sulfite and sulfide standards are not suitable for such analysis, as the recovery of analyte was in the range of 5–50%. The quantitative recovery of sulfur from all CRMs (in the range of 93–103%) with the repeatability of 4.1– 5.0% was obtained using DL-cysteine as calibration standard. Slightly worse results (recoveries in the range of 89–107% and 90–108%) were
Table 3 Comparative results of analysis of biological certified reference materials using different calibration standard solutions by the proposed HR-CS FMAS method (CS rotational line: 258.055 nm, 3 pixels, n = 3). Calibration standard used
Soya bean flour INCT-SBF-4a
Mixed polish herbs INCT-MPH-2b
Corn flour INCT-CF-3c
Recovery of S ± SD, %
RSD, Recovery of % S ± SD, %
RSD, Recovery of % S ± SD, %
RSD, %
DL-cysteine
111.5 104.7 107.4 10.0 13.2 103.3
1.2 1.4 1.4 0.9 5.9 4.1
3.7 3.4 3.5 0.4 0.2 3.8
4.1 3.9 3.9 5.0 4.1 4.1
100.0 101.6 103.3 6.3 50.3 102.1
5.0 4.5 4.6 6.9 2.5 5.0
L-cystine
108.0 ± 1.5 1.4
90.0 ± 3.4
3.8
105.9 ± 4.7 4.4
H2SO4 Na2SO4 (NH4)2SO4 Na2SO3 Na2S
a b c
± ± ± ± ± ±
1.4 1.5 1.6 0.1 0.8 4.3
90.5 86.9 88.9 7.4 4.9 92.9
−1
Certified value: 4245 ± 471 mg kg . Certified value: 2410 ± 140 mg kg−1. Certified value: 919 ± 121 mg kg−1.
± ± ± ± ± ±
± ± ± ± ± ±
5.0 4.6 4.8 0.4 1.3 5.1
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Fig. 2. Influence of the possible matrix constituents of analyzed samples on the CS molecular absorbance.
obtained for (NH4)2SO4 and L-cystine standards with the repeatability of 1.4–4.6%. On the basis of these results the DL-cysteine standard was chosen for quantitative analysis.
3.3. Method characteristics and application The method of determination of sulfur by HR CS FMAS using DLcysteine calibration standard was characterized (data presented in Supplementary Table S1 (Appendix A)). The obtained limits of detection (10.9 mg L− 1) and quantification (36.4 mg L− 1) are lower in comparison to the values obtained by others, as can be seen in Table 1. The repeatability of results is better than 5.8%. The accuracy of the method was checked by the determination of sulfur in certified reference materials (soya bean flour, corn flour, mixed herbs). The obtained results were consistent with the certified values (Table 3). The method was also applied for the determination of various sulfur contents in diluted biological samples, such as milk, egg white, white and red wine, mineral water as well as in bitter mineral water contain–1 + (4700 mg L–1) and ing large amounts of HCO− 3 (1210 mg L ), Na 2+ –1 Mg (2900 mg L ) ions. In order to check the occurrence of possible interferences the concentration of sulfur in mineral waters and wines was determined by standard addition and external calibration graph techniques. Good comparability of the results obtained by both calibration techniques was obtained as it is shown in Table 4. The recovery experiment was also performed. The recovery of sulfur added to these samples was in the range of 97–105%. The results obtained for both mineral waters were also consistent (difference of 1–2%) with the values declared by producers (see Table 4). The method can be applied for determination of analyte in samples containing low (mineral water) and high (mineral bitter water) concentrations of sulfur.
The examination of possible matrix interferences during the determination of sulfur in egg white was performed by comparison of results obtained for samples diluted 10, 20 and 40-fold with 0.1 mol L−1 NaCl. The consistent results (the difference below 5%) were obtained for analysis of all these samples (Table 4). A similar experiment was performed for milk samples. The concentration of sulfur was determined in undiluted milk and milk 3-fold diluted with 0.1 mol L−1 HCl. Also in this case the consistent results were obtained. The results of analysis are in agreement with literature data too [27,41].
4. Conclusions The present work shows that the HR CS FMAS technique using a commercially available spectrometer can be employed for the direct determination of total sulfur in food and beverage samples. Same samples in particular can be analyzed after minimal pre-treatment step, just simple dilution. The developed procedure for sulfur determination by the formation and measurement of CS molecules in air–acetylene flame is simple and more sensitive in comparison to current procedures. It was found that rotational line of the molecule, number of pixels and type of standard solution of sulfur strongly affected the sensitivity of the method. The lowest LOD for sulfur determination was obtained for the measurements of absorption of CS molecule using the rotational line at 258.055 nm with the wavelength range covering 3 pixels (central pixel ± 1) and DL-cysteine in 0.2 mol L−1 HNO3 solution as calibration standard. The best accuracy of results was also obtained using organic DL-cysteine standard as was confirmed by analysis of biological CRMs. The obtained data suggests, that biological matrix, containing high content of carbon, might cause better efficiency of formation and/or better thermal stability of CS molecule. The developed method can be applied
Table 4 Results of analysis of sulfur in food samples by the proposed HR-CS FMAS method (CS rotational line: 258.055 nm, 3 pixels, calibration standard: DL-cysteine; n = 5). Sample
Mineral water(a) Red wine White wine Mineral bitter water(b) Fresh cow's milk Chicken egg white Tomato leaves NIST-SRM1573a(c) (a) (b) (c)
Sample dilution factor
1 1 1 10 3 1 10 20 40 0.5 g of sample, digested, evaporated and diluted to 10 mL
Content of sulfur ± SD
Recovery of S ± SD after addition of 318 mg L−1 sulfur
Calibration graph technique
Standard addition technique
31.9 ± 0.9 mg L−1 82.3 ± 2.4 mg L−1 260 ± 4 mg L−1 6900 ± 100 mg L−1 194 ± 10 mg kg−1 197 ± 9 mg kg−1 2190 ± 30 mg kg−1 2140 ± 130 mg kg−1 2070 ± 50 mg kg−1 9580 ± 430 mg kg -1
31.8 ± 0.4 mg L−1 79.1 ± 3.3 mg L−1 252 ± 4 mg L−1 7050 ± 120 mg L−1 –
100 ± 1% 99 ± 2% 97 ± 3% 105 ± 2% –
–
–
–
–
Concentration of sulfates declared by the producer: 95.0 mg L−1 as SO2− (31.6 mg L−1 as S). 4 Concentration of sulfates declared by the producer: 21,200 ± 2110 mg L−1 as SO2− (7060 ± 700 mg L−1 as S). 4 Digested certified reference material: information content of sulfur: 9600 mg kg−1.
E. Zambrzycka, B. Godlewska-Żyłkiewicz / Spectrochimica Acta Part B 101 (2014) 234–239
for sulfur determination in fresh and mineralized food and beverage samples containing various amounts of analyte and complex matrix. Additionally due to its simplicity, low reagent consumption, and elimination of the use and generation of hazardous substances, the proposed method matches the principles of “green chemistry”. Acknowledgments The authors thank the Meranco Aparatura Kontrolno-Pomiarowa i Laboratoryjna Sp. z.o.o. for making possible the carrying out of measurements with the ContrAA 300 spectrometer. E. Zambrzycka is a beneficiary of the project “Scholarships for PhD students of Podlaskie Voivodeship” co-financed by the European Social Fund, the Polish Government and Podlaskie Voivodeship. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.sab.2014.08.041. References [1] N. Jakubowski, N. Mihalopoulos, S. Mann, W.D. Lehmann, Speciation of sulfur, in: R. Cornelis, H. Crews, J. Caruso, K.G. Heumann (Eds.), Handbook Of Elemental Speciation II, Species in the Environment, Food, Medicine & Occupational Health, John Wiley & Sons, Ltd., England, 2005, pp. 378–407. [2] M.E. Nimni, B. Han, F. Cordoba, Are we getting enough sulfur in our diet? Nutr. Metab. 4 (2007) 24–38. [3] M. Masters, R.A. McCance, The sulphur content of foods, Biochem. J. 33 (1939) 1304–1312. [4] C.J. Mussinan, M.E. Keelan, Sulfur compounds in foods. An overview, ACS Symposium Series, American Chemical Society, Washington, DC, 1994, pp. 1–6. [5] Methylsulfonylmethane (MSM), monograph, Altern. Med. Rev. 8 (2003) 438–441. [6] N. Jackowetz, E. Li, R. Mira de Orduña, Sulphur dioxide content of wines: the role of winemaking and carbonyl compounds, Res. Focus 3 (2011) 1–7. [7] B.J. Freedman, Sulphur dioxide in foods and beverages: its use as a preservative and its effect on asthma, Br. J. Dis. Chest 74 (1980) 128–134. [8] Regulation of the Minister of Health of 20 April 2010 r. (Dz.U. 2010 Nr 72 poz. 466). (In Polish). [9] C.G. Kowalenko, C.J. Van Laerhoven, Total sulfur determination in plant tissue, in: Y. P. Kalra (Ed.), Handbook of Reference Methods for Plant Analysis, CRC Press LLC, United States of America, 1998, pp. 93–103. [10] S.M.B. Brienza, R.P. Sartini, J.A. Gomes Neto, E.A.G. Zagatto, Crystal seeding in flow-injection turbidimetry: determination of total sulfur in plants, Anal. Chim. Acta. 308 (1995) 269–274. [11] Z. Kowalewska, H. Laskowska, Comparison and critical evaluation of analytical performance of wavelength dispersive X-ray fluorescence and ultraviolet fluorescence for sulfur determination in modern automotive fuels, biofuels, and biocomponents, Energy Fuel 26 (2012) 6843–6853. [12] M. Necemer, P. Kump, M. Rajcevic, R. Jacimovic, B. Budic, M. Ponikvar, Determination of sulfur and chlorine in fodder by X-ray fluorescence spectral analysis and comparison with other analytical methods, Spectrochim. Acta Part B 58 (2003) 1367–1373. [13] S.S.M. Hassan, M.S.A. Hamza, A.H.K. Mohamed, A novel spectrophotometric method for batch and flow injection determination of sulfite in beverages, Anal. Chim. Acta. 570 (2006) 232–239. [14] M. Koch, R. Koppen, D. Siegel, A. Witt, I. Nehls, Determination of total sulfite in wine by ion chromatography after in-sample oxidation, J. Agric. Food Chem. 58 (2010) 9463–9467. [15] R.E. Santelli, E.P. Oliveira, M. de Fátima, B. de Carvalho, M.A. Bezerra, A.S. Freire, Total sulfur determination in gasoline, kerosene and diesel fuel using inductively coupled plasma optical emission spectrometry after direct sample introduction as detergent emulsions, Spectrochim. Acta Part B 63 (2008) 800–804. [16] E. Paredes, M.S. Prats, S.E. Maestre, J.L. Todolí, Rapid analytical method for the determination of organic and inorganic species in tomato samples through HPLC– ICP-AES coupling, Food Chem. 111 (2008) 469–475. [17] J. Wang, H. Yi, C. He, H. Li, Sample preparation method for the determination of total sulfur in plant materials, Commun. Soil Sci. Plant Anal. 30 (1999) 599–603. [18] R.S. Amais, G.L. Donati, J.A. Nobrega, Application of the interference standard method for the determination of sulfur, manganese and iron in foods by inductively coupled plasma mass spectrometry, Anal. Chim. Acta. 706 (2011) 223–229.
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Florez, Direct determination of sulfur in solid samples by means of high-resolution continuum source graphite furnace molecular absorption spectrometry using palladium nanoparticles as chemical modifier, J. Anal. At. Spectrom. 27 (2012) 401–412. [24] B. Welz, H. Becker-Ross, S. Florek, U. Heitmann, M.G.R. Vale, High-resolution continuum-source atomic absorption spectrometry — what can we expect? J. Braz. Chem. Soc. 14 (2003) 220–229. [25] B. Welz, D.L.G. Borges, F.G. Lepri, M.G.R. Vale, U. Heitmann, High-resolution continuum source electrothermal atomic absorption spectrometry — an analytical and diagnostic tool for trace analysis, Spectrochim. Acta Part B 62 (2007) 873–883. [26] U. Heitmann, H. Becker-Ross, S. Florek, M.D. Huang, M. Okruss, Determination of non-metals via molecular absorption using high resolution continuum source absorption spectrometry and graphite furnace atomization, J. Anal. At. Spectrom. 21 (2006) 1314–1320. [27] M.D. Huang, H. Becker-Ross, S. Florek, U. Heitmann, M. Okruss, C.D. Patz, Determination of sulfur forms in wine including free and total sulfur dioxide based on molecular absorption of carbon monosulfide in the air–acetylene flame, Anal. Bioanal. Chem. 390 (2008) 361–367. [28] M.D. Huang, H. Becker-Ross, S. Florek, U. Heitmann, M. Okruss, Determination of sulfur by molecular absorption of carbon monosulfide using a high-resolution continuum source absorption spectrometer and an air–acetylene flame, Spectrochim. Acta Part B 61 (2006) 181–188. [29] M.D. Huang, H. Becker-Ross, S. Florek, U. Heitmann, M. Okruss, Direct determination of total sulfur in wine using a continuum-source atomic-absorption spectrometer and an air–acetylene flame, Anal. Bioanal. Chem. 382 (2005) 1877–1881. [30] S.R. Oliveira, J.A. Gomes Neto, J.A. Nóbrega, B.T. 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Determination of sulfur in food by high resolution continuum source flame molecular absorption spectrometry☆ Elżbieta Zambrzycka, Beata Godlewska-Żyłkiewicz ⁎ Institute of Chemistry, University of Bialystok, Hurtowa 1, 15-399 Białystok, Poland
a r t i c l e
i n f o
Article history: Received 15 May 2014 Accepted 21 August 2014 Available online 10 September 2014 Keywords: Molecular absorption spectrometry Sulfur determination Food analysis
a b s t r a c t In the present work, a fast, simple and sensitive analytical method for determination of sulfur in food and beverages by high resolution continuum source flame molecular absorption spectrometry was developed. The determination was performed via molecular absorption of carbon monosulfide, CS. Different CS rotational lines (257.959 nm, 258.033 nm, 258.055 nm), number of pixels and types of standard solution of sulfur, namely: sulfuric acid, sodium sulfate, ammonium sulfate, sodium sulfite, sodium sulfide, DL-cysteine, and L-cystine, were studied in terms of sensitivity, repeatability of results as well as limit of detection and limit of quantification. The best results were obtained for measurements of absorption of the CS molecule at 258.055 nm at the wavelength range covering 3 pixels and DL-cysteine in 0.2 mol L−1 HNO3 solution as a calibration standard. Under optimized conditions the limit of detection and the limit of quantification achieved for sulfur were 10.9 mg L−1 and 36.4 mg L−1, respectively. The repeatability of the results expressed as relative standard deviation was typically b 5%. The accuracy of the method was tested by analysis of digested biological certified reference materials (soya bean flour, corn flour and herbs) and recovery experiment for beverage samples with added known amount of sulfur standard. The recovery of analyte from such samples was in the range of 93–105% with the repeatability in the range of 4.1–5.0%. The developed method was applied for the determination of sulfur in milk (194 ± 10 mg kg− 1), egg white (2188 ± 29 mg kg− 1), mineral water (31.0 ± 0.9 mg L− 1), white wine (260 ± 4 mg L− 1) and red wine (82 ± 2 mg L−1), as well as in sample rich in ions, such as bitter mineral water (6900 ± 100 mg L− 1). © 2014 Elsevier B.V. All rights reserved.
1. Introduction Sulfur, next to oxygen and silicon, is the most abundant constituent of minerals. Sulfur is also one of the major elements essential to life, as many functionally important cellular components, such as essential amino acids, contain this element [1]. Sulfur is mostly taken from food rich in proteins, such as eggs, fresh milk, red meat and fish [2]. There are also many vegetables containing high content of sulfur like sweet corn, spinach, onion and garlic and cruciferous vegetables (broccoli, cauliflower, cabbage, kale) (0.7–14 g kg−1) [3,4]. In mineral and drinking waters sulfur is mainly present as sulfates and sulfides, while in food of animal origin (chicken eggs, meat, milk) as sulfur-containing amino acids: methionine, cysteine, cystine and taurine. A variety of fruit, vegetables, and grains naturally include sulfur as organic compound — methylsulfonylmethane, also known as dimethyl sulfone [5]. Sulfur is often added to food as a preservative. Sulfur dioxide and sulfites, which
☆ Selected paper from the European Symposium on Atomic Spectrometry ESAS 2014 & 15th Czech–Slovak Spectroscopic Conference, Prague, Czech Republic, 16–21 March 2014. ⁎ Corresponding author. Tel.: +48 85 745782; fax: +48 85 7470113. E-mail address: [email protected] (B. Godlewska-Żyłkiewicz).
http://dx.doi.org/10.1016/j.sab.2014.08.041 0584-8547/© 2014 Elsevier B.V. All rights reserved.
release sulfur dioxide in acidic solution, are chemical additives used for inhibiting undesirable bacterial growth and oxidation processes in wine production [6,7]. The maximum permitted amount of sulfur in wine and drinking water in various countries is strictly regulated by legislation concerning food safety. The maximum contaminant level of sulfate in drinking water in Poland is 250 mg L−1, while the maximum limit of sulfides is only 0.05 mg L−1[8]. In the European Union the maximum limits for the total SO2 in red and white wines are 150 and 200 mg L−1 (dry wine), 200 and 250 mg L−1 (medium wine) and 300 and 400 mg L−1 (sweet wine), respectively [6]. The determination of sulfur is of interest in many areas such as human health, agriculture and food industries [9]. Among the classical methods of sulfur determination are gravimetric analysis based on precipitation of barium sulfate and combustion to sulfur dioxide with detection by titration, thermal conductivity, flow-injection turbidimetry [10], and ultraviolet fluorescence spectrometry [11]. Other methods used include wavelength dispersive X-ray fluorescence spectrometry [11,12], spectrophotometry [13], ion chromatography [14], inductively coupled plasma optical emission spectrometry [15–17] and inductively coupled plasma mass spectrometry [18,19]. Nevertheless, each of these methods has its own methodological limitations, such as poor precision and selectivity resulting from a complex chemistry of this element.
E. Zambrzycka, B. Godlewska-Żyłkiewicz / Spectrochimica Acta Part B 101 (2014) 234–239
The development of high resolution continuum source atomic absorption spectrometry (HR CS AAS) introduced new possibilities in the determination of sulfur and other non-metals such as phosphorus and halogens (Cl, I, Br) based on absorption spectra of formed molecules [20–26]. The first application of the HR CS AAS instrument for the determination of sulfur in wine was done in flame by Huang et al. [27–29]. The studies performed using different continuum source spectrometers developed at the ISAS Berlin [28,29] have shown, that in the spectral range between 200 and 465 nm several molecular bands can be found. It was proved that the spectra band around 258 nm appears due to the presence of carbon monosulfide (CS) molecular absorption, whereas at around 325 nm it appears due to the absorption of the SH molecule. Since CS is a more stable molecule than SH (the bond enthalpies of C–S and S–H bonds are equal to 714.1 and 344.3 kJ mol−1), the molecular absorption of CS is mostly used for sulfur determination. During the last decade a number of papers reporting the determination of sulfur based on CS spectra using commercially available HR CS AAS spectrometers equipped with a flame [30–34] or graphite furnace [34–38] have been published. The volatility of various sulfur chemical forms is highly variable, hence careful optimization of flame conditions, chemical modifiers and furnace temperature as well as type of sulfur standard is essential for accurate results of analysis. Quantitative analysis was performed by standard addition [26,27,29,34] or external calibration graph technique [31,34,35,37]. Different inorganic compounds containing sulfur were tested as standards, such as H2SO4[32], Na2SO4[32], MgSO4·7H2O [30,33,38], (NH4)2SO4[31], Na2SO3[29], and Na2S [35]. Among them sulfates were preferred for determination of CS in flame, as they are stable under various conditions (acidity, content of oxidizing agents, ethanol, etc.) (see Table 1) [28]. Kowalewska [34] tested various organic compounds, dibuthyl sulfide ((C4H9)2S), tertnonyl polysulfide ((C6H13C(CH3)2)2Sx), carbon disulfide (CS2), as standards for the determination of sulfur in petroleum products by HR CS FMAS and HR CS GFMAS, but the accurate results were only obtained using heavy petroleum products with known sulfur content for calibration. Other organic sulfur compounds, such as thiourea [36], and L-cysteine [38] were used as standards during the determination of sulfur by slurry [39] and solid sampling [35,36,38] HR GF MAS. The thiourea standard in the presence of Pd [36] modifier was employed for sulfur determination in biological samples, while L -cysteine standard was used for its determination in coal [38]. It was found that Lcysteine behavior during the pyrolysis process was similar to the behavior of the coal sample [38]. All of these methods were used for the analysis of samples containing sulfur at the levels of 0.03–6%. The aim of this work was to develop a simple method suitable for the determination of sulfur in food/beverage samples by HR CS FMAS after minimal sample pre-treatment (dilution with appropriate solvent).
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The type of standard solution suitable for such analysis was selected. The influence of matrix constituents on the analytical signal of different standards was investigated. During optimization of experimental parameters the best conditions in terms of sensitivity, repeatability of results, limit of detection and limit of quantification, as well as accuracy of results were always selected. 2. Experimental 2.1. Instrumentation and flame conditions All the absorption measurements were performed using a high resolution continuum source atomic absorption spectrometer ContrAA® 300 (Analytik Jena AG, Jena, Germany) equipped with a continuum light source — xenon short-arc lamp XBO 301 (GLE, Berlin, Germany) with the arc in a hot spot mode suitable for all elements determination, a double monochromator consisting of a prism pre-monochromator and a high resolution echelle grating monochromator and a charge-coupled device (CCD) array detector with 588 pixels. The detector allows for the visualization of the environment of the analytical line using 200 pixels. An air-acetylene flame was used for the determination of sulfur via CS molecule under optimized conditions (burner height: 8 mm, burner length: 100 mm, air–C2H2 flow rate: 120 L h−1). The digestion of samples was performed in a microwave system 150 Ethos Plus (Milestone, Italy). 2.2. Reagents and analytical solutions All reagents used were of analytical grade or higher. Sulfur standard solutions were prepared by dissolution of sulfuric acid (95%, POCH, Poland), high purity ammonium sulfate anhydrous (POCH, Poland), sodium sulfate anhydrous (Sigma-Aldrich, USA), sodium sulfite anhydrous (POCH, Poland), sodium sulfide non-hydrate (Sigma-Aldrich, USA) in Milli-Q water obtained using a Milli-Q system (Millipore, USA). L-Cystine (Sigma-Aldrich, USA) and DL-cysteine (Sigma-Aldrich, USA) were dissolved in 0.2 mol L−1 HNO3 (69.5%, Trace Select, Fluka, France). Hydrochloric acid (37%, fuming, Trace Select, Fluka, France) and sodium chloride (anhydrous, Sigma-Aldrich, USA) were used for milk and chicken egg white preparation. Nitric acid (69.5%, Trace Select, Fluka, France) and hydrogen peroxide (30%, Merck, Germany) were used for the digestion of plant samples. Sodium hydrogen carbonate (POCH, Poland), ethanol (99.8%, POCh, Poland), lead(II), calcium and magnesium nitrates (POCH, Poland) were used to study the matrix interference. The chicken eggs, pasteurized cow's milk with 0.5% milk fat, Hungarian bitter water “Hunyadi Janos”, mineral water “Saguaro”, Hungarian semi-dry white wine “Tokaj Furmint”, Bulgarian semi-dry red wine “Sophia Sakar”, samples of which were investigated in this
Table 1 Literature data on sulfur determination based on molecular absorption of CS using HR CS FMAS. Wavelength Number of pixels
Calibration standard
258.055 nm
3
DL-cysteine, L-cystine,
H2SO4, Na2SO4, (NH4)2SO4, Na2SO3, Na2S
258.056 nm 257.593 nm 258.055 nm
3 5
(NH4)2SO4, H2SO4, KHSO4, Na2SO3, (NH4)2S, Na2S2O3 MgSO4·7H2O
257.595 257.958 258.056 258.056
nm nm nm nm
1
(NH4)2SO4
5
257.595 nm
5
H2SO4,Na2SO4, (NH4)2SO4, Na2S2O8 MgSO4·7H2O
258.056 nm 11
Petroleum products, (C4H9)2S, CS2, (C6H13C(CH3)2)2Sx
Limit of detection
RSD
Samples
10.9 mg L−1 b5.8% Chicken egg white; fresh milk; wine; bitter and mineral water; CRM: soya bean flour (INCT-SBF-4); mixed polish herbs (INCT-MPH-2); corn flour (INCT-CF-3); tomato leaves (NIST-1573a) 2.4 mg L−1 – CRM: cast iron (EURONOM ZRM 428-2); cast iron 4.4 mg L−1 – (EURONOM ZRM 484-1) 22.4 mg L−1 b5.7% Orange leaves; CRM: tomato leaves (NIST 1573a); peach leaves (NIST 1547); spinach (NIST 1570a) 15.1 mg L−1 4.8% Fungicide, fertilizer −1 22.4 mg L 5.7% 21.8 mg L−1 4.2% 100 mg L−1 b9.7% Coal, CRM: coal (NIST 1632b) 34.7 mg L−1 – 21 mg L
−1
–
In bold — calibration standard used for quantitative analysis; RSD - relative standard deviation.
Ref. This paper [28] [30] [31]
[32]
Medicinal plants; CRM: spinach leaves (NIST 1570a); apple leaves [33] (NIST 1515) Crude oil; petroleum products; CRM: residual fuel oil (1634b, 1618, JPI); [34] fuel oil (F007, F009)
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study, were locally available brands, collected in supermarkets in Białystok (Poland). The accuracy of the HR CS FMAS method was checked by analyzing certified reference materials (CRM) of soya bean flour (INCT-SBF-4), mixed polish herbs (INCT-MPH-2), corn flour (INCT-CF-3) supplied by the Institute of Nuclear Chemistry and Technology (Warsaw, Poland) and tomato leaves (NIST-SRM-1573a) supplied by the National Institute of Standards and Technology (Gaithersburg, Maryland, USA).
2.3. Analytical procedure The rotational line at 258.055 nm was chosen for measurements of absorption of the CS molecule. For the evaluation of the absorption signals, the integrated absorbance of three pixels (central pixel ± 1), which covers a range of approximately 6 pm, was used. All absorbance values are mean values based on three repetitive measurements. The dynamic background correction technique with reference was used. The quantitative determination of sulfur was carried out by the external calibration graph technique based on DL-cysteine standard. During the optimization of each parameter the best conditions in terms of sensitivity, repeatability of results as well as limit of detection were always selected. Limits of detection (LOD) and quantification (LOQ) were calculated according to following equations: LOD = 3SDblank / a, LOQ = 10SDblank / a, where “SDblank” is standard deviation of the absorbance measurements of blank solution (0.2 mol L− 1 HNO3) and “a” is a slope of the calibration graph.
2.4. Preparation of samples The egg white was prepared by a modified procedure developed by Awadé and Efstathiou [40]. The egg white was manually separated from the egg yolk, introduced to plastic vessel (90 mL) and weighed. Next, the egg white was diluted 1:1 with an equivalent amount of 0.1 mol L−1 NaCl and homogenized with a magnetic stirrer for 3 h. For the absorbance measurements 1 g (~ 1 mL) aliquots of obtained solutions were transferred to smaller plastic vessels (20 mL) and diluted with 0.1 mol L−1 NaCl to 5 mL, 10 mL or 20 mL. Prior to analysis the 1.0 g samples of pasteurized cow's milk were diluted to 3 mL with 0.1 mol L− 1 HCl and then homogenized with a magnetic stirrer for 5 min. The samples of waters and wine were acidified with HNO3 (to obtain final acid concentration of 0.2 mol L− 1) and next degassed under a vacuum for 1 h. All certified reference materials were digested before analysis in a closed microwave system. About 0.5 g each of the CRM plant materials was placed into a Teflon vessel and then 6 mL of concentrated HNO3 and 1 mL of H2O2 were added. The heating program included the following steps (power/temperature/hold time): step 1: 250 W/ 110 °C/2 min; step 2: 0 W/110 °C/2 min; step 3: 250 W/185 °C/6 min; step 4: 400 W/185 °C/5 min; and step 5: 600 W/185 °C/5 min. The obtained solutions were transferred to quartz crucibles, evaporated near to dryness and next diluted with 0.2 mol L−1 HNO3 to 10 mL.
3.1. Optimization of signal evaluation mode The absorption spectrum of the CS molecule produced in an air– acetylene flame registered around 258 nm under optimized burner parameters is presented in Fig. 1. This band shows several sharp rotational lines, among which the strongest lines were observed at 257.959 nm, 258.033 nm and 258.055 nm. Different rotational lines were used for the determination of sulfur by other authors [21,28,30–35] because they vary in the sensitivity and the possible risk of occurrence of interferences from e.g. Fe [26,28] or Co [23]. Additionally, by taking more CS lines into account, i.e., summing up the integrated absorbance of individual rotational lines, the sensitivity as well as the signal-to-noise ratio could be significantly improved as was reported in [21,26,33]. In this work the calibration graphs for (NH4)2SO4 standard were constructed on the basis of absorbance signals registered for each individual line at CP (central pixel) and for the sum of absorbances of three most sensitive lines (at 257.959, 258.033 and 258.055 nm). The data are shown in Table 2. The sensitivity of measurements for individual lines was similar, but the lowest limit of detection was obtained for measurements at 258.055 nm. The sensitivity of measurements based on the sum of absorbances increased by a factor of 3, while the LOD value was from 2 to 5 times lower than these calculated for individual lines (Table 2). These results are in agreement with the results obtained by Heitmann et al. [26] and Bechlin et al. [33]. Such positive effect was observed for summing of the most sensitive lines. However, the summing of more than 3 absorption lines, which differ in shape, neighborhood and sensitivity, improved the sensitivity of measurements in a lesser extent. It was found that for the summing of 6 and 9 lines the sensitivity increased by factors of 5 and 6.8 in comparison to the measurements of one individual line, but the limit of detection was higher due to the higher noise level. For practical purposes only the single line at 258.055 nm was chosen for sulfur determination in further work. The sensitivity of measurements may be improved by increasing the number of pixels used for detection. The influence of wavelength integrated absorbance on the sensitivity and precision was studied by varying the number of pixels used for detection from 1 to 7. The slopes and correlation coefficients of the calibration graphs, limits of detection, limits of quantification and repeatability for different number of pixels are summarized in Table 2. It was observed that the sensitivity of measurements when 5 pixels were taken for signal evaluation was 2.9 times higher in comparison to that obtained for the evaluation of 1 pixel and 20% higher than for the evaluation of 3 pixels. However, the LOD value calculated using 5 pixels was worse than that obtained using 3 pixels. Using 7 pixels the sensitivity was similar to that obtained for 5 pixels,
3. Results and discussion The method for sulfur determination in food is based on the measurements of the absorption band of the stable diatomic CS molecule by HR CS FMAS. The formation of the CS molecule in a flame is promoted by using an excess of acetylene, which produces a reducing environment of the flame. The initial optimization experiments were performed using (NH4)2SO4 as sulfur standard. The obtained data were always analyzed in terms of sensitivity, repeatability (calculated as RSD), limit of detection (LOD) and limit of quantification (LOQ) values.
Fig. 1. Absorption spectrum of the CS molecule with the strongest rotational lines: 1) 258.055 nm, 2) 258.033 nm and 3) 257.959 nm, obtained for a DL-cysteine solution containing 600 mg L −1 of S.
E. Zambrzycka, B. Godlewska-Żyłkiewicz / Spectrochimica Acta Part B 101 (2014) 234–239 Table 2 Influence of the CS rotational line, summation of absorbance signals of rotational lines (Σ lines) and number of pixels on the sensitivity, linear correlation coefficient of calibration graph (R), limit of detection (LOD), limit of quantification (LOQ) obtained for (NH4)2SO4 as sulfur standard (n = 3). Wavelength
Number of pixels
Slope
R
LOD
LOQ −1
mg L 257.959 258.033 258.055 Σ lines 258.055
nm nm nm nm
1 1 1 1 3 5 7
1.4·10−5 1.4·10−5 1.5·10−5 4.3·10−5 3.6·10−5 4.4·10−5 4.3·10−5
0.9993 0.9998 0.9998 0.9998 0.9998 0.9989 0.9998
27.8 31.3 12.0 5.6 10.0 12.9 10.9
92.8 104.3 40.0 18.8 33.3 42.9 36.3
but the noise that is computed to the total integrated absorbance increased (relative standard deviation of measurements reached 17%). For further studies the absorbance values at 258.055 nm based on the detection of 3 pixels were used, due to good sensitivity, precision and the lowest LOD value. The limit of detection for sulfate standard (10.0 mg L− 1) under optimized measurement conditions is lower in comparison to the values reported in the literature (Table 1). 3.2. Standard solutions for calibration It was observed that the sensitivity of the CS molecular absorption produced in the air–acetylene flame depends on the chemical form of the sulfur-containing substances [28,34]. As was mentioned earlier different standards were tested for quantitative analysis of sulfur, but irrespective of the type of analyzed samples, mainly sulfates were chosen for calibration purposes (Table 1). The properly optimized analytical procedure should provide conditions for achieving proper stabilization of sulfur in flame regardless of the chemical form in which sulfur is present in the analyzed samples. This aspect was investigated in more detail in the current work. Initially the influence of different concentrations of nitric acid (in the range of 0.01–1 mol L− 1) on the CS molecular absorbance obtained using different sulfur containing standards, namely sulfuric acid, sodium sulfate, ammonium sulfate, sodium sulfite, sodium sulfide, DL-cysteine, and L-cystine, was studied. It must be noticed that due to low solubility of L-cystine in water it must be dissolved in acidic conditions. Addition of 0.04 mol L− 1 HNO3 to aqueous solution of L-cystine caused 40% increase of absorbance of sulfur. Further acidification of this standard solutions caused only a slight increase of absorbance. The absorbance of DL-cysteine and Na2SO4, after addition of 0.01 mol L−1 HNO3 increased by 20% in comparison with aqueous standard. The absorbance of H2SO4 and (NH4)2SO4 solutions remained constant in the studied range of HNO3 concentration. The effect of the rapid increase of absorbance for sulfite (1000%) and sulfide (400%) standards in the presence of nitric acid at a concentration above 0.04 mol L−1 was observed, which was also reported by Huang et al. [27,28]. In order to avoid the differences in the formation of CS molecule from standards and digested samples all solutions were further prepared in 0.2 mol L−1 HNO3. The parameters of the calibration graphs using different sulfur containing compounds, limits of detection, limits of quantification and repeatability for different standard concentrations (data presented in Supplementary Table S1 (Appendix A)) were analyzed. The highest sensitivity of the calibration graphs was obtained using solutions containing sulfur in the form of sulfite. Huang el al. [28] explained that effect as the generation of gaseous SO2 during sample nebulization and the more efficient transport into the flame, as compared with the ionic forms of sulfur. However, the sulfites present originally in samples might be converted into sulfates when the medium contains oxidizing compounds (e.g. during sample digestion step with HNO3 and H2O2) or into SO2 if it is very acidic or alkaline. Hence, this standard is not recommended for analysis of digested samples. Sodium sulfide in acidic solution is
237
converted into volatile H2S, which is easily transferred into flame. It was observed that, during storage of such solutions gaseous H2S is continuously removed from the analyzed samples. After 15 min of storage the signal of sulfur decreased by 25%, while after 40 min it decreased by almost 80%. For these reasons, although the highest sensitivities and lowest limits of detection were obtained for Na2SO3 and Na2S solutions, these compounds are not suitable as standards for the determination of sulfur in real biological samples by HR CS FMAS (see Table 3). Baysal and Akman [32] have found the best sensitivity and wide linear range of the concentration graph (0.005–20% of S) using sulfuric acid as a standard. However, the increasing concentration of sulfuric acid in standards causes a rapid decreasing of solution pH, which may change the conditions of the formation of CS molecule. For this reason we excluded this standard from further investigations. Similar sensitivities as well as limits of detection were obtained for all other sulfur standards, but the best repeatability of results was observed for DL-cysteine and (NH4)2SO4 standards. The influence of the possible matrix constituents of analyzed samples on the CS molecular absorbance was examined and is shown in Fig. 2. The absorbance of organic standards of sulfur (DL-cysteine, L-cystine) remained practically constant (differences b 5%) in the presence of 10% −1 hydrogen peroxide, 10% ethanol, 70 mg L−1 HCO− 3 and 0.1 mol L NaCl. Greater effects were observed for (NH4)2SO4 standard solution (differences in the range of 5–15%). The high resolution of applied spectrometer significantly minimizes the possibility of spectral interferences from foreign metals, but does not eliminate the occurrence of nonspectral interferences. The lowest influence of Pb2+, Mg2+ and Ca2+ ions was observed on the signal of DL-cysteine. This suggests, that a biological matrix, containing high content of carbon, might be responsible for better formation and better thermal stability of the CS molecule. Kowalewska [34] has also observed positive effect of organic matrix (heavy petroleum products) on the efficiency of CS formation in flame. Similar effect was also found in graphite furnace by Ferreira et al. [36] during analysis of biological samples and Kowalewska [34] during analysis of heavy petroleum products. During the analysis of coal samples losses of sulfur from standard based on MgSO4, due to the formation of less stable competitive molecule in the presence of coal carbon, were reported, therefore L-cysteine standard was applied for such analysis [38]. Different standards were used for the determination of sulfur in CRMs of biological samples (Table 3). It was confirmed that sulfite and sulfide standards are not suitable for such analysis, as the recovery of analyte was in the range of 5–50%. The quantitative recovery of sulfur from all CRMs (in the range of 93–103%) with the repeatability of 4.1– 5.0% was obtained using DL-cysteine as calibration standard. Slightly worse results (recoveries in the range of 89–107% and 90–108%) were
Table 3 Comparative results of analysis of biological certified reference materials using different calibration standard solutions by the proposed HR-CS FMAS method (CS rotational line: 258.055 nm, 3 pixels, n = 3). Calibration standard used
Soya bean flour INCT-SBF-4a
Mixed polish herbs INCT-MPH-2b
Corn flour INCT-CF-3c
Recovery of S ± SD, %
RSD, Recovery of % S ± SD, %
RSD, Recovery of % S ± SD, %
RSD, %
DL-cysteine
111.5 104.7 107.4 10.0 13.2 103.3
1.2 1.4 1.4 0.9 5.9 4.1
3.7 3.4 3.5 0.4 0.2 3.8
4.1 3.9 3.9 5.0 4.1 4.1
100.0 101.6 103.3 6.3 50.3 102.1
5.0 4.5 4.6 6.9 2.5 5.0
L-cystine
108.0 ± 1.5 1.4
90.0 ± 3.4
3.8
105.9 ± 4.7 4.4
H2SO4 Na2SO4 (NH4)2SO4 Na2SO3 Na2S
a b c
± ± ± ± ± ±
1.4 1.5 1.6 0.1 0.8 4.3
90.5 86.9 88.9 7.4 4.9 92.9
−1
Certified value: 4245 ± 471 mg kg . Certified value: 2410 ± 140 mg kg−1. Certified value: 919 ± 121 mg kg−1.
± ± ± ± ± ±
± ± ± ± ± ±
5.0 4.6 4.8 0.4 1.3 5.1
238
E. Zambrzycka, B. Godlewska-Żyłkiewicz / Spectrochimica Acta Part B 101 (2014) 234–239
Fig. 2. Influence of the possible matrix constituents of analyzed samples on the CS molecular absorbance.
obtained for (NH4)2SO4 and L-cystine standards with the repeatability of 1.4–4.6%. On the basis of these results the DL-cysteine standard was chosen for quantitative analysis.
3.3. Method characteristics and application The method of determination of sulfur by HR CS FMAS using DLcysteine calibration standard was characterized (data presented in Supplementary Table S1 (Appendix A)). The obtained limits of detection (10.9 mg L− 1) and quantification (36.4 mg L− 1) are lower in comparison to the values obtained by others, as can be seen in Table 1. The repeatability of results is better than 5.8%. The accuracy of the method was checked by the determination of sulfur in certified reference materials (soya bean flour, corn flour, mixed herbs). The obtained results were consistent with the certified values (Table 3). The method was also applied for the determination of various sulfur contents in diluted biological samples, such as milk, egg white, white and red wine, mineral water as well as in bitter mineral water contain–1 + (4700 mg L–1) and ing large amounts of HCO− 3 (1210 mg L ), Na 2+ –1 Mg (2900 mg L ) ions. In order to check the occurrence of possible interferences the concentration of sulfur in mineral waters and wines was determined by standard addition and external calibration graph techniques. Good comparability of the results obtained by both calibration techniques was obtained as it is shown in Table 4. The recovery experiment was also performed. The recovery of sulfur added to these samples was in the range of 97–105%. The results obtained for both mineral waters were also consistent (difference of 1–2%) with the values declared by producers (see Table 4). The method can be applied for determination of analyte in samples containing low (mineral water) and high (mineral bitter water) concentrations of sulfur.
The examination of possible matrix interferences during the determination of sulfur in egg white was performed by comparison of results obtained for samples diluted 10, 20 and 40-fold with 0.1 mol L−1 NaCl. The consistent results (the difference below 5%) were obtained for analysis of all these samples (Table 4). A similar experiment was performed for milk samples. The concentration of sulfur was determined in undiluted milk and milk 3-fold diluted with 0.1 mol L−1 HCl. Also in this case the consistent results were obtained. The results of analysis are in agreement with literature data too [27,41].
4. Conclusions The present work shows that the HR CS FMAS technique using a commercially available spectrometer can be employed for the direct determination of total sulfur in food and beverage samples. Same samples in particular can be analyzed after minimal pre-treatment step, just simple dilution. The developed procedure for sulfur determination by the formation and measurement of CS molecules in air–acetylene flame is simple and more sensitive in comparison to current procedures. It was found that rotational line of the molecule, number of pixels and type of standard solution of sulfur strongly affected the sensitivity of the method. The lowest LOD for sulfur determination was obtained for the measurements of absorption of CS molecule using the rotational line at 258.055 nm with the wavelength range covering 3 pixels (central pixel ± 1) and DL-cysteine in 0.2 mol L−1 HNO3 solution as calibration standard. The best accuracy of results was also obtained using organic DL-cysteine standard as was confirmed by analysis of biological CRMs. The obtained data suggests, that biological matrix, containing high content of carbon, might cause better efficiency of formation and/or better thermal stability of CS molecule. The developed method can be applied
Table 4 Results of analysis of sulfur in food samples by the proposed HR-CS FMAS method (CS rotational line: 258.055 nm, 3 pixels, calibration standard: DL-cysteine; n = 5). Sample
Mineral water(a) Red wine White wine Mineral bitter water(b) Fresh cow's milk Chicken egg white Tomato leaves NIST-SRM1573a(c) (a) (b) (c)
Sample dilution factor
1 1 1 10 3 1 10 20 40 0.5 g of sample, digested, evaporated and diluted to 10 mL
Content of sulfur ± SD
Recovery of S ± SD after addition of 318 mg L−1 sulfur
Calibration graph technique
Standard addition technique
31.9 ± 0.9 mg L−1 82.3 ± 2.4 mg L−1 260 ± 4 mg L−1 6900 ± 100 mg L−1 194 ± 10 mg kg−1 197 ± 9 mg kg−1 2190 ± 30 mg kg−1 2140 ± 130 mg kg−1 2070 ± 50 mg kg−1 9580 ± 430 mg kg -1
31.8 ± 0.4 mg L−1 79.1 ± 3.3 mg L−1 252 ± 4 mg L−1 7050 ± 120 mg L−1 –
100 ± 1% 99 ± 2% 97 ± 3% 105 ± 2% –
–
–
–
–
Concentration of sulfates declared by the producer: 95.0 mg L−1 as SO2− (31.6 mg L−1 as S). 4 Concentration of sulfates declared by the producer: 21,200 ± 2110 mg L−1 as SO2− (7060 ± 700 mg L−1 as S). 4 Digested certified reference material: information content of sulfur: 9600 mg kg−1.
E. Zambrzycka, B. Godlewska-Żyłkiewicz / Spectrochimica Acta Part B 101 (2014) 234–239
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