Abstract: Chronopotentiometric stripping analysis is proposed for selenium determination in feed. Experimental conditions of the technique were investigated and optimised. As a working electrode served mercury film deposited at glassy carbon. Accuracy of the method was confirmed by analysing the standard reference material-wheat durum flour. Detection limit obtained for proposed technique was 0.5 μg dm-3, while limit of quantitation was 1 μg dm-3. Developed method was used for selenium determination in fish meal, meat-bouny meal, sows feed and yeast.
Introduction
Selenium is naturally occuring element essential for humans, animals, plants and microorganisms. Many substances express both toxic and positive effect depending on its amount and chemical form, but for selenium is specific that the ratio of toxic to essential dose is very narrow.
Biochemical and metabolic role of selenium is rather complex. Selenium forms an integral part of the enzimes glutathione-peroxidase (Rotruck et al., 1973), iodothyronine deiodinase, thyoredoxin reductase (Berry et al., 1991), metalloproteines, fatty and binding proteins (Bansal et al., 1989), selenoproteines P and W (Read et al., 1990) and many other proteins.
The inhibition of the toxic effects of heavy metals such as arsenic, cadmium, mercury and tin is due to the formation of their stable selenides (Ganther, 1978).
Selenium deficiency causes a number of specific degenerative diseases in livestock associated with the growth and reproduction (Oldfield, 1997). In food animals and poultry, low selenium intake is associated with a wide range of practical and costly problems including both male and female infertility, low immune system of animals and disorders of thyroide hormone metabolism (Whitehair and Miller, 1985). In Table 1 are presented selenium-related problems in food animal production.
The distribution of selenium in the soil varies widely. For example, the contents of selenium in some soils of Ireland reaches 1250 mg kg-1, while the soil of the certain areas of New Zeland contain less than 0.1 mg kg-1 of selenium. As a result the selenium content of plant-derived feed ingredients is difficult to predict. Research in selenium content of such samples requires measurement of widely different selenium concentrations in many types of feed with different origin. To be useful, a method for selenium determination needs to work equally well in any type of the sample and must be sensitive enough to measure the very lowest levels encountered. Knowing the exact selenium content in feed is necessary because of positive as well as toxic effects of the element.
| Table 1: | Problems in food animal production related to selenium defficiency |
| aMarin-Guzman and Mahan (1989), bMaas and Koller (1985), cPatterson et al. (1957), dEdens (1998), eWakebe (1998) | |
On the basis of this data selenium supplementation is performed if necessary. Nowadays, feed supplementation with selenium has become common way of modern meat production, influencing not only animals health but, actually, aiming food products with higher selenium content and better quality. In Korea the production of pork meat enriched with selenium is well known. Similar programme is being performed with poultry meat. New Zeland, which is largely selenium deficient has a history now of over 35 years of selenium use in agriculture, with enormous benefits to animal production and human health.
The low content of selenium in biological samples demands highly sensitive analytical methods for its determination. A lot of methods have been developed for the determination of selenium. Some of them involve the reaction between selenium (IV) and aromatic diamine to obtain a piazselenol, which is determined by fluorimetry (Mejuto-Marti et al., 1987; Lu and Zheng, 1991). Modern methods in order to perform the speciation of selenium compounds combine some chromatographic technique with specific and selective detector. Separation techniques that can be used are gas chromatography (Measures and Burton, 1980; Al-Attar and Nickless, 1990; Salenko et al., 1991) or high performance liquid chromatography (Gilon et al., 1995; Gomez-Ariza et al., 2002; Angeles et al., 2000). Separated compounds are subsequently determined by inductively coupled plasma atomic emission (ICP AES) (Narasaki et al., 2000) or mass spectrommetry (ICP MS) (Darrouzes et al., 2005; Feldmann et al., 2000), atomic spectrommetry after electrotermal atomisation (Gilon et al., 1995) or after hydride generation (Sun et al., 2002; Gomez-Ariza et al., 2002). These techniques require very sophisticated and expensive instrumentation as well as exploatation. There application is complex and time consuming. Simpler and less expensive techniques that can be used for selenium determination are stripping techniques. Sensitivity of all stripping techniques is atributed to the preconcentration step that is performed prior analytical one. The aim of the preconcentration step is to concentrate the analyte in the electrode medium. The dissolution of the deposit can be performed in different ways providing the qualitative and quantitative date about the analyte. In voltametric stripping techniques the dissolution is performed by the potential swep and in chronopotentiometric stripping techniques by the constant current. In cathodic stripping voltammetry selenium is accumulated at the mercury electrode surface by the electroreduction either in the form of mercury-selenide (Batley, 1986; Companella et al., 1989; Pottin-Gautier et al., 1995) or by the formation of the intermetallic compounds such as copper-selenium (Van den Berg and Khan, 1990; Matsson et al., 1995) and rhodium-selenium (Wang and Lu, 1993). Selenium can also be preconcentrated by the adsorptive collection in the form of complex with 3,3`-diaminobenzidine on a hanging mercury drop electrode (Stara and Kopanica, 1998). Anodic stripping voltammetry can be applied only if the solid working elecrodes are used, for example gold electrode (Tan and Kounaves, 1998). Besides stripping voltammetry stripping techniques that have been applied for selenium determination with detection limits down to μg dm-3 levels are reductive Potentiometric Stripping Analysis (PSA) (Christensen et al., 1980) and Chronopotentiometric Stripping Analysis (CSA). Chronopotentiometric stripping analysis has been rarely applied for selenium determination. Method was used for selenium determination in urine and plasma samples (Gozzo et al., 1999) and in flow system for the determination in muscle and bovine liver (Eskilsson and Haraldsson, 1987). In CSA minor charging currents and more accurate time measurement comparing to current measurement in voltametric techniques makes the CSA determinations more selective and accurate. Considering all advantages of the technique, the aim of this study was to define simple and inexpensive technique which can be used for sensitive determinations of selenium in natural samples. After the optimisation of the sample pretreatment developed method was applied for selenium determination in different types of feed, such as fish meal, meat-bouny meal, sows feed and yeast.
Materials and Methods
Instrumentation
Investigation was performed in laboratory for Instrumental Methods of Analysis,
Department for Applied and Engineering Chemistry at the Faculty of Technology.
Chronopotentiometric stripping analysis was performed using the computerised
system for electrochemical stripping analysis of our own construction (Suturović
et al., 1992; Suturović, 1996). Mercury film electrode was used
as a working electrode. Mercury was deposited at the glassy carbon by a constant
current (50 μA) electrolysis from the separate solution containing 100
mg dm-3 of mercury(II) and 0.02 mol dm-3 of hydrochloric
acid. Before each deposition of the mercury glassy carbon was washed with acetone
and double distilled water. As a counter electrode served platinum wire (n =
0.7 mm, l = 7 mm) and the reference was Ag/AgCl, KCl (3.5 mol dm-3)
electrode.
Chemicals And Vessels
All chemicals used in this study were of ultra pure grade (Supra pure).
For all dilutions and dissolutions triply distilled water was used. All vessels
and cells were washed with nitric acid (1:1), distilled and triply distilled
water.
Solutions
Selenium (IV) stock solution (2 g dm-3) was prepared by dissolving
sodium-selenite pentahydrate (p.a., Merck, min 99% of Na2SeO3·5H2O,
iodometric) in 0.1 mol dm-3 hydrochloric acid and kept in polyethylene
bottle in dark. Working solutions of selenium (IV) were prepared by diluting
selenium stock solution with triply distilled water. Nitrogen used for the deaeration
of the analysed solution was of extra purity.
Reference Material
Wheat durum flour with the certified selenium content of 1.23±0.09
mg kg-1 was used as a reference material (RM 8436) for the verification
of the method accuracy. Prior analysis the material was dried at 85°C for
4 h (1 cm layer).
Sample Pretreatment
Sample (3 g) was transferred to a quartz long-necked flask and 10 cm3
of nitric and 6 cm3 of perchloric acid were added. After evolution
of dark nitrogen-oxides fumes 6 cm3 (6x1 cm3) of nitric
acid was added aiming the complete decomposition, i.e., to obtain clear and
colourless solution. The obtained solution was completely evaporated. Perchloric
acid fumes were collected with the glass tube connected to a vacuum-pump. After
cooling, dry residue was treated with 1.5 cm3 of 5 mol dm-3
hydrochloric acid and heated for 20 min at 120°C in order to reduce Se(VI)
to Se(IV), since only Se(IV) is electroactive. Finally, solution was filtered
and transferred to a 25 cm3 glass flask with triply distilled water.
CSA Analysis
Prepared sample solution (20 cm3) was deaerated by passing nitrogen
for 600 sec. During the deaeration time working electrode was held in triply
distilled water in order to avoid the damage of the electrode surface with nitrogen
bubbles. After the deaeration the electrode was returned into the solution and
electrolysis was performed during 600 sec at a potential of –0.1 V. Stirring
rate was 4000 rpm. After the rest period of 10 sec the stripping step was performed
by the reduction current. Because of a different sample matrix, dissolution
currents varied. For the sample of sows feed dissolution current was 10.2 μA,
while currents of 13.3 μA; 17.1 μA and 18.6 μA were used for
the samples of fish meal, meat-bony meal and yeast, respectively. All samples
were analysed in five probes. Selenium content was calculated by the method
of a calibration curve. In blank solutions selenium was not detected.
Accuracy Test
Samples of the reference material were analysed after the wet acid digestion
and reduction of Se(VI) as described above. The electrolysis time (t) was 180
sec and the applied dissolution current was 1.8 μA.
Results and Discussion
Influence Of The Electrode Thickness
Mercury film thickness (d) influences directly at the amount of the mercury-selenide
formed and therefore at the methods sensitivity. Influence of the mercury film
thickness on selenium dissolution time (τ) was examined by varying the
electrolysis time from 60 sec (~31 nm) to 420 sec (~226 nm). Solutions containing
60 μg dm-3 of selenium (IV) were used for the examination. Applied
electrolysis potential was –0.15 V, electrolysis time 60 sec dissolution
current 3 μA and stirring rate 4000 rpm. According to the reproducibility
and height of the analytical signal (Table 2), electrolysis
time of 300 sec was accepted as an adequate and corresponded to ~163 nm film
thickness (presuming 100% electrolysis efficiency). In Table 2
are also shown the values of standard deviation (S) and coefficient of variation
(CV) calculated on the basis of five analysis. Mercury film depositet under
this conditions enabled 15-20 analysis.
Influence Of The Supporting Electrolyte Concentration
The role of supporting electrolyte is to provide the conductivity of the
solution, to adjust pH value and to minimise the migration current. In this
study as a supporting electrolyte was used hydrochloric acid. Influence of its
concentration was investigated in the range 0.1-1 mol dm-3.
| Table 2: | Influence of the mercury film thickness on selenium analytical signal |
| Table 3: | Influence of the dissolution current on selenium analytical signal |
| Table 4: | Determined selenium contents in feed |
Although the concentration of 0.2 mol dm-3 did not provide highest sensitivity it was chosen as optimal, because it enabled sharp, well-defined signal and its good repeatability.
Influence Of The Electrolysis Potential On Selenium Analytical Signal
In order to obtain sharp and well-defined dissolution signal of mercury-selenide,
determination of optimal electrolysis potential is of a great importance. Electrolysis
potential examined was in the range from -0.05 to -0.3 V. Solutions containig
20 μg dm-3 of Se (IV) were implied in the investigation. Dissolution
time for each potential value shown in Fig. 1 represents average
value of five analyses on the same mercury film. Dissolution time decreased
with more negative potential, probably because of less amount of Hg2+
ions available for the reaction with selenium, since mercury solubility is higher
at more anodic potentials. Highest analytical signal of selenium was obtained
applying electrolysis potential of –0.05 V, but with poor reproducibility.
Best reproducibility was obtained applying electrolysis potentials of –0.1
V and -0.15 V. As an optimal electrolysis potential was chosen -0.1 V.
Influence of the Electrolysis Time on Selenium Analytical Signal
Influence of the electrolysis time on selenium analytical signal was investigated
in standard solutions containing 10 μg dm-3 of selenium (IV).
Electrolysis time varied from 60 sec to 300 sec. Dissolution current was 1.8
μA. Longer electrolysis time contributed to the higher sensitivity, because
it corresponded to higher selenium concentrations in the electrode medium.
Influence Of The Dissolution Current
Dissolution current is one of the most important experimental conditions
of chronopotentiometric stripping analysis. Its influence was investigated in
solutions containig 10 μg dm-3 of selenium (IV) and varied from
1.5 to 2.6 μA (Table 3). Each shown value of the analytical
signal represents average value of five analyses on different mercury films.
In case when analytical signal corresponding to certain current value represented
average value of five analyses on the same mercury film, the dependence could
not be defined, although the reproducibility was much more satisfactory. Electrolysis
time was 90 sec.
| Fig. 1: | Dependence of the selenium analytical signal on the electrolysis potential |
With the decrease of the dissolution current selenium analytical signal increased approximately exponentially. Dissolution currents higher than 2.6 μA caused the significant decrease of the sensitivity, while currents lower than 1.5 μA were insufficient for the dissolution of the deposit and caused the extension of the chronopotentiogram and problems in the determination of the inflection point.
Linearity
The dependence of selenium analytical signal on the content (Cm)
was defined for the range 20-60 μg dm-3. Electrolysis time was
90 sec and reducing current was 1.8 μA. In the examined range experimental
results were in correlation (r = 0.9947) with the assumed linear dependence
(τ = 0.0301Cm + 0.84; n = 5). Obtained results are in agreement
with the results of the examination of the influence of the electrolysis time,
because longer electrolysis time in stripping techniques is analoguos with higher
concentration of the analyte. Even though the dependence was linear method of
the standard addition which is simpler and which compensate the matrix influence
could not be applied due to the significant τ intercept. For the calculation
of the concentration the method of calibration plot was used.
Repeatability (Analytical Reproducibility)
After the optimisation of the experimental conditions repeatability was
determined by seven consequent analysis at the same mercury film. Solution containing
10 μg dm-3 of Se(IV) was analysed applying electrolysis time
of 90 sec and dissolution current of 1.5 μA. Qualitative analysis of selenium
was based on the dissolution potential shown in the first column (Fig.
2).
| Fig. 2: | Repeatability of the selenium determination |
Reproducibility of the dissolution potential was very good and expressed as variation coefficient was 0.32%. Quantitaion was performed according to the dissolution time shown in second and third columns in seconds and the intern units of the analyser (1 sec = 81.37 units), respectively. Repeatability expressed as a coefficient of variation was 6.02%.
Detection Limit And Limit Of Quantitation
After the optimisation of the CSA parameters the detection limit (LOD) of
0.5 μg dm-3 was obtained for the electrolysis time of 600 sec
and the quantitation limit (LOQ) of 1 μg dm-3 for same electrolysis
time. The method of calibration plot was used for the determination of LOD and
LOQ (ACS Committee on Environmental Improvement, 1980).
Accuracy Test
The method accuracy was confirmed by analysing standard reference material
in five probes. All determined contents were in the range 1.23±0.09 mg
kg-1, which is certified for the standard reference material RM 8436.
Selenium Determination In Feed
As can be seen in Table 4 selenium content in all samples
of feed was below 0.2 mg kg-1. According to the Serbian regulations
(Regulation book, 2000) minimal selenium content recommended for sows feed is
0.1 mg kg-1. Determined content in analysed sample was 10% lower.
Taking into account that fish meal and meat-bony meal are added in the premixes
in 5-7 %, while yeast is added in 2-3 %, it is obvious that these ingredients
can not be considered as selenium source in feed, where selenium content should
be depending on the type of the feed in the range from 0.1 to 0.45 mg kg-1.
On the basis of these results it can be concluded that selenium supplementation
in feed produced from these ingredients is required.
Conclusions
Simple and inexpensive technique for chronopotentiometric stripping analysis of selenium was defined. The sensitivity of the defined method enables the determination of low levels of selenium present in biological samples. The correctness and the accuracy of the sample pretreatment procedure were confirmed with the good results of recovery assay. Methods accuracy was confirmed by analysing the standard reference material. Besides methods applicability for selenium determination in feed, it can be assumed that the method can also be applied with success for selenium determination in various premixes where expected contents of selenium are higher or for analysis of different biological samples.