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CHAPTER 7
SPECTROPHOTOMETRIC DETERMINATION OF SELENIUM IN
ENVIRONMENTAL, BIOLOGICAL AND PHARMACEUTICAL SAMPLES
7.1 INTRODUCTION
7.2 ANALYTICAL CHEMISTRY
7.3 APPARATUS
7.4 REAGENTS AND SOLUTIONS
7.5 PROCEDURES
7.6 RESULTS AND DISCUSSION
7.7 APPLICATIONS
7.8 CONCLUSIONS
7.9 REFERENCES
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7.1 INTRODUCTION
Selenium was discovered by Berzelius and Gahn in 1817. It is widely present
in nature in relatively small concentrations in rocks, plants, coal and other fossil fuels.
Selenium is comparatively rare and its abundance [1] in the lithosphere is 9×10-6 %.
The important minerals containing selenium are clausthalite PbS , crookesite
(Cu,Tl,Ag)2Se, eucairite (Cu,Ag)2Se, naumannite (Ag,Pb)Se. Selenium is also present
in the soil in certain areas of the U.S.A (the dry plains of Dakota, Wyoming and
Kansas) and is taken up by vegetation which then becomes poisonous to animals;
their meat is then rendered unfit for human consumption. Selenium is however, an
essential trace element in some animal diets.
Selenium is one of the trace element which plays an active role in many
biological systems [2] as it has toxicological and physiological effects [3,4]. Selenium
compounds are extensively used in paints, dyes, glass, electricals, rubber, insecticides,
industries [5] and photocell devices [6] in which variations in the frequencies of the
incident light cause a corresponding variation in the electric current. Grey crystalline
selenium is the only allotrope suitable for this purpose, its conductivity increases
approximately thousand fold when illuminated. Gray selenium is used to a greater
extent for rectification, utilizing the property of asymmetric conduction exhibited by
thin layers of this allotrope [7]. Less important uses of selenium are in the
manufacture of colored (red or reddish-yellow) glasses or ceramic and enamel
pigments. Both selenium and tellurium have been used as secondary vulcanizing
agents for natural rubber in the form of organo-compounds and as oxidation inhibitors
in lubricating oils [8].
Very pure selenium is obtained by heating the crude material in hydrogen at
650o to form hydrogen selenide, which is then passed through a silica tube at 1000o to
decompose [9]. Hydrogen sulfide is more stable to heat than the selenide and it
passes out of the system unchanged. The hydrides of elements which are less stable
to heat than hydrogen selenide, are not formed at 650oC. It is apparent from vapour
density determinations that Se8 molecules are present below 550oC. The vapour is
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yellow at the boiling (685oC) and it dissociates into Se6, Se2 (above 900oC) molecules
and to atomic selenium occurs with the increase in the temperature. A mass
spectrometric study [10] of selenium vapour has provided evidence for the existence
of Se4 and Se7 molecules in the vapour and the enthalpies of vaporization have been
measured for each of these species.
The determination of selenium is of considerable interest because of its
contrasting biological effects. Selenium is a toxic element as well as a trace element
present in animals and humans. High concentration of selenium causes pulmonary
edema, abdominal pain, jaundice, chronic gastrointestinal diseases, hair loss and
fatigue in humans [11] and its deficiency causes Keshan and Kaschin Beck diseases in
humans, which are frequently reported in China [12]. It also plays a major role in the
life cycle of plants (Cruceferae family), which absorb organoselenium compounds
accumulated in the soils of semiarid areas and may poison livestock that graze on
them. Selenium enters into natural water through seepage from seleniferrous soil and
industrial waste. Water drained from such soil may cause severe environmental
pollution and wild life toxicity. Selenium is also reported to be present in cigarette
paper, tobacco [13] and various cosmetic samples [14].
Selenium is a trace mineral that is essential to good health but required only in
small amounts [15,16]. Selenium is incorporated into proteins to make selenoproteins,
which are important antioxidant enzymes. The anti-oxidant properties of
selenoproteins help to prevent cellular damage from free radicals. Free radicals are
natural by-products of oxygen metabolism that may contribute to the development of
chronic diseases such as cancer and heart disease [16,17]. Other selenoproteins help
to regulate thyroid function and play a role in the immune system [18-20]. Because of
its ant-ioxidant role, selenium has been studied for its potential to protect the body
from many degenerative diseases, including Parkinson’s and cancer. Selenium is
thought to protect cells against cancer because a form of selenium from yeast was
found to have caused cancer cells in test tubes and in animals to undergo apoptosis or
programmed cell death. Selenium is found in some meats and seafood. Animals that
eat grains or plants those were grown in selenium-rich soil have higher levels of
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selenium in their muscle. In U.S., meats and bread are common sources of dietary
selenium [21,22]. Some nuts are also sources of selenium.
Some industrial and agricultural processes release selenium as a byproduct and
selenium from such sources has caused environmental disaster [23]. Selenium is also
a semiconductor and is used in some types of solid-state electronics as well as in
rectifiers [24], it is an essential nutrient at trace level but toxic in excess [25]. The
threshold limit value for selenium compounds in air is 0.1- 0.2 mgL-1 and in water it is
4.0 ppm [26].
The toxicity, availability and environmental mobility of selenium are very
much dependent on its chemical forms [27]. Selenium can occur in different oxidation
states in organic and inorganic forms. In many environmental matrixes, e.g. natural
water, soils, etc. the predominant oxidation states of selenium are Se(IV) and Se(VI).
Precise knowledge of the amounts of selenium and its compounds present in a system
is therefore required for accurate assessment of the environmental and biological
impact of selenium. This has resulted in an increasing need for analytical methods
suitable for their determination at trace levels.
7.2 ANALYTICAL CHEMISTRY
Selenium is widely spread in relatively small concentrations in rocks, plants,
coal and other fossil fuels. Owing to the importance of selenium, several analytical
techniques have been reported for the determination of selenium [28-31].
Russell et al. described the determination of selenium and tellurium in crude
silver chloride produced as a by-product of the refining of gold and in high-purity
uranium oxide [32]. Selenium and tellurium were separated from one another as well
as from the numerous other substances in the sample before they were extracted into
organic solvents and determined spectrophotometrically. For the determination of
selenium 3,3'-diaminobenzidine was used and for tellurium, diphenylthiourea was
190
used. A minimum of 2 ppm of each element was determined in silver chloride and
0.3 ppm in uranium oxide.
Langmyhr and Omang described a spectrophotometric determination of
selenium(IV) with 1,1�-dianthrimide [33] The method was based on the reaction of
selenium with 1,1�-dianthrimide in concentrated sulfuric acid, which formed a
������ �� � ���� �� �� ��� �� � ������ ��� ����� � ��� ��� ��� ��� ���
complex formation was utilized for spectrophotometric determination of up to 0.2 mg
of Se in 25 mL. Boron, germanium, tellurium, bromide and fluoride were interfered.
Langmyhr and Myhrstad described the complex formation in concentrated
sulfuric acid between selenium(IV) and 1,1'-dianthrimide (DA) by spectrophotometry,
infrared spectroscopy and chemical analysis [34]. The system was found to contain
two species, a Se2DA complex and a selenium-1,2,7,8-diphthaloylcarbazole complex.
Langmyhr and Dahl reported an investigation of the applicability of
2,2'-dianthrimide in spectrophotometry and the determination of selenium(IV) [35].
2,2'-Dianthrimide was studied as an analytical reagent and compared with the
properties of 1,1'-dianthrimide, while 1,1'-dianthrimide reacted with B, Ge, Se and Te,
2,2'-dianthrimide was found to react only with selenium(IV). A straight line
������� ������������� ������� ���� �!������������� ���� ��������� ����
but the value of 2,2'-dianthrimide as a reagent for selenium(IV) was reduced by the
high absorption of the reagent.
Kawashima and Ueno reported a spectrophotometric determination of trace
amounts of selenium in iron and steel with 4-methyl-o-phenylenediamine [36]. Brown
reported a spectrophotometric determination of selenium(IV) with diaminochrysazine
[37]. Kasterka reported N-"#-hydroxypropyl)–o-phenylenediamine and
N-methyl-o-phenylenediamine as reagents for the spectrophotometric determination
of selenium [38,39].
Neve et al. described three important techniques for decomposition of organic
materials for differential determination of the selenium oxidation states [40]. The
method applied to vegetable and biological samples. The only method that was found
191
suitable for the selective determination in aqueous samples gave unsatisfactory results
for organic materials, the recovery of both native and added selenium was very low.
The methods were critically discussed and a procedure was recommended for the
accurate determination of total selenium in organic samples.
Idriss et al. reported a spectrophotometric and potentiometric studies to the
reaction of selenium(IV) with eosin and 1,10-phenanthroline in aqueous solution [41].
An ion association ternary complex with a stoichiometric ratio
selenium-(phenanthroline)2-(eosin)2 was formed. The stability constant of the
complex was determined and the optimum conditions for the spectrophotometric
determination of Se(IV) was established.
Campbell and Yahaya described a spectrophotometric determination of
selenium with dithizone [42]. Microgram amounts of selenium(IV) were determined
by the decrease in absorbance of dithizone in carbon tetrachloride solution at 620 nm.
$��� ���� ����������� ����!������������ ����� ��%��������� �!��������"&'(
were 0.6 % and 0.4 % respec ����)�*�����"� ������ �����(�������"� �����-
� �����(������ ��!�����
Neve et al. described an atomic absorption spectrophotometric determination
of ultramicro amounts of selenium in sulphuric acid medium [43]. Selenium(IV) was
determined after the extraction into toluene with an aromatic o-diamine and the
addition of nickel(II) prior to atomization. In the studied samples, total selenium
(0.003–0.022 µg of selenium in 1 mL sulfuric acid) was present only in the tetravalent
state. The detection limit of the method was 0.003 µg of selenium.
Bhat and Gupta described a reagent system consisting of 4-nitrophenyl
hydrazine and 8-quinolnol for the photometric determination of selenium [44].
4-Nitrophenyl hydrazine was oxidized with selenious acid in 6M hydrochloric acid to
4-nitrophenyldiazonium chloride which was then coupled with 8-quinolnol, which
formed a purple colored azoxine dye in alkaline medium with an absorption
maximum at 550 nm. The molar absorptivity and Sandell’s sensitivity were
3.2×104 Lmol+�cm+� ��� ����%� � ��-2 respectively. The method applied for the
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detection and determination of complex materials such as cabbage leaf and cigarette
paper.
Bodini and Alzamora described a spectrophotometric determination of trace
amounts of selenium with 4,5,6-triaminopyrimidine(TAP) [45]. In this method TAP
reacted in acidic aqueous medium with selenium(IV), which formed a piazselenol
with an absorption maximum at 362 nm with a molar absorptivity of 1.72×104
Lmol+�cm+�. The compound was stable but not extracted into non-polar solvents. The
calibration graph was linear up to 10 ppm of selenium, with a detection limit of
0.1 ppm in the sample solutions. Of the many different ions tested only iron (III)
(in the presence of chloride) and tin (II) interfered. The method produced good
reproducibility with a relative standard deviation of 1.5 % for pure solutions. The
method applied to the analysis of water and electrolytic copper. Lavale and Dave
described a spectrophotometric determination of selenium with chromotropic acid
[46].
Kasterka described a spectrophotometric determination of selenium with
2-aminodiphenylamine in an acidic medium [47]. The optimum hydrogen ion
concentration ranges from about 0.1 to 5M. The molar absorptivity at λ=352 nm was
1.81×104 Lmol–1cm–1. The product was extracted as an ion-association complex with
perchlorate into a mixture of hexanol and chlorobenzene. The kinetics of the reaction
was investigated.
Bodini et al. reported a reagent 5,5-dimethyl-1,3-cyclohexanedione which
reacted in dilute acid solution with selenium(IV), which formed a benzoxaselenol and
showed an absorption maximum at 313 nm [48]. The molar absorptivity of the
method was 4.0×103 Lmol-1cm-1. The calibration graph was linear up to 30 ppm of
selenium, with a detection limit of 0.1 ppm in the final solutions.
Kasterka reported the condensation reactions of Se(IV) with
3,4-diaminobenzoic acid and 4-bromo-1,2-phenylenediamine by means of UV spectra
and kinetic investigations [49]. A mechanism for the formation of
1,2,3-benzoselenadiazole in acidic medium was proposed. The influence of
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substitution at C4 in 1,2-phenylenediamine on the reactivity of the system was
discussed.
Manish et al. reported a simple and sensitive method for the
spectrophotometric determination of selenium(IV) using 6-amino-1-naphthol-3-
sulphonic acid as a reagent [50]. The molar absorptivity and Sandell’s sensitivity of
the method were found to be 18.5×103 Lmol-1cm-1 and 0.004 μgcm-2 respectively.
Beer’s law was obeyed in the concentration range of 0.03-0.3 μgL-1 of selenium.
Safavi and Afkhami reported a highly sensitive catalytic spectrophotometric
method for the determination of selenium(IV) and selenium(VI) [51]. The method
was based on the catalytic effect of Se(IV) in redox reaction of bromate with
semicarbazide in hydrochloric acid media. The determination range of both analyses
was 50-4000 ngmL-1. Selenium as low as 4.7 ngmL-1 was determined by this method.
The application of the method to the determination of selenium in Kjeldahl tablets and
in a health-care product was described.
Ramachandran and Kumar described a reaction of selenium with
2,3-diaminonaphthalene which was reinvestigated with bromide ion as a catalyst [52].
In acid medium, selenium reacted with the above reagent, which formed a complex
extracted with cyclohexane and with an absorption maximum at 378 nm. The molar
absorptivity of the complex was 17.5×103 Lmol-1cm-1. Beer’s law was obeyed in the
concentration range of 0.5-�%� ,-1 of selenium.
Pyrzynska developed the conditions for a spectrophotometric determination of
selenium with 1-naphthyloamine-7-sulfonic acid (Cleve�� ����( -�./� 0�����um(IV)
formed a yellow complex with this ligand in sulfuric acid media with maximum
absorbance at 350 nm. The molar absorptivity was 8.9×104 Lmol-1cm-1. The
������� ������������������� �.��� �!���������� �0������������� ��� )����
�����1� ��-2. The amount of Se in a column of unit cross-sectional area with the
absorbance of 0.001. The interference of various ions was studied. The method was
applied for the determination of selenium in a vitamin supplement.
194
Agrawal et al. reported a reagent system for the spectrophotometric
determination of selenium in environmental and cosmetic samples using leucocrystal
violet(LCV) [54]. The method was based on the reaction of selenium with acidified
potassium iodide to liberate iodine, which oxidized LCV to crystal violet with an
absorption maximum at 593 nm. Beer's law was obeyed over the concentration range
of 0.5-���� �!�����������!�����������!%��,����������� ����������� ��� )
and Sandell's sensitivity were found to be 3.68×105 Lmol-1cm-1 a�� �����% � ��-2
respectively.
Mousavi et al. reported a simple and sensitive flow infection
spectrophotometric method for the determination of selenium [55]. The method was
based on the catalytic effect of Se(IV) on the reduction reaction of thionin with
sulphide ion, monitored spectrophotometrically at 598 nm. Beer’s law obeyed in the
range 0.005-1.5 μgmL-1 of selenium. The detection limit was 5 μgmL-1. The relative
standard deviation for eight replicate measurements was 1.1% for 1 μgmL-1 of
selenium.
Varadarajan et al. reported bis(ethanedithioamido)-2,4-dioxo-3-
oxyminopentane, bis(EDA)DOP as a sensitive and selective reagent for the
spectrophotometric determination of total selenium traces [56]. The method was
based on the color reaction between selenium(IV) and bis(EDA)DOP on heating the
mixture at 50°C for 2.0 minutes which is extracted in 1-octanol from an acidic
medium with respect to 2-3 M HCl on shaking for 1.5 minutes. The absorbance of
the extracted species was measured at 495.0 nm and the molar absorptivity was
1.268×104 Lmol-1cm-1. The complex system obeyed Beer’s law within
0.2-15.0 mgmL-1 in Ringbom’s optimum working range of 4.36-12.02 µgmL-1 with a
sensitivity of 2.21 ngcm-2 for effective spectrophotometric determination of total
selenium. The method was applied to the determination of total selenium in various
synthetic mixtures and other samples.
Melwanki and Seetharamappa described spectrophotometric determination of
selenium(IV) using methdilazine hydrochloride as a reagent [57]. The reagent formed
195
a red radical cation by selenium (IV) acid medium and exhibited an absorption
maximum at 513 nm. Beer’s law was valid over the concentration range 0.1-2.3
mgL-1 of selenium(IV). Sandell’s sensitivity of the reaction was found to be
3.57 ngcm-2 and the molar extinction coefficient was 9.32×104 Lmol-1cm-1.
Revanasiddappa and Kiran Kumar reported a direct method for the
spectrophotometric determination of micro amounts of selenium(IV) using variamine
blue as a chromogenic reagent [58]. The method was based on the reaction of
selenium with potassium iodide in an acidic medium to liberate iodine, which
oxidized variamine blue to a violet colored species with an absorption maximum at
546 nm. Beer’s law was obeyed in the range 2-20 μgmL-1 of selenium in a final
volume of 10 mL. The molar absorptivity and Sandell’s sensitivity for the colored
system were found to be 2.6×104 Lmol-1cm-1 and 0.003 μgcm-2 respectively.
Revanasiddappa and Kiran Kumar reported used thionin as a reagent for the
spectrophotometric determination of selenium(IV) in real samples of water, soil, plant
materials, human hair, synthetic cosmetics and in pharmaceutical preparations [59].
The molar absorptivity and Sandell’s sensitivity of the method were found to be
7.33×104 Lmol-1cm-1 and 0.0011 μgcm-2 respectively. Beer’s law was obeyed in the
range 1.0-5.0 μgmL-1 of selenium in a final volume of 10 mL.
Gurkan and Akcay developed a simple and sensitive catalytic
spectrophotometric method for the determination of trace amounts of selenium [60].
The method was based on the catalytic effect of Se(IV) on the reduction of maxilon
blue-SG by sodium sulfide. Indicator reaction was followed spectrophotometrically
by measuring an absorption maximum at 654 nm. Selenium could quantitatively be
determined in the range 0.004-0.200 μgmL-1 Se(IV) with a detection limit of
0.205 ngmL-1 selenium(IV).
Narayana et al. described a rapid and sensitive spectrophotometric method for
the determination of trace amounts of selenium using starch and iodine as
chromogenic reagents [61]. The proposed method was based on the reaction of
selenium with potassium iodide in an acidic medium to liberate iodine. This reacted
196
with starch to form a blue colored species with an absorption maximum of 570 nm.
Beer's law was obeyed in the range of 2-�%� �!����������������������� ��� )
and Sandell's sensitivity were found to be 1.40×104 Lmol-1cm-1 and 5.45×10-3� ��-2
respectively. The proposed method was successfully applied to determine selenium in
a sample of natural water, polluted water, soil sludge, biological samples and human
hair. Guo et al. described a new vapor generation technique utilizing UV irradiation
coupled with atomic absorption for the determination of selenium in aqueous
solutions [62].
Ningli reported a spectrophotometric method for the determination of trace of
selenium. The method was based on the bromopyrogallol red oxidized fading reaction
by selenium(IV) in neutral solution [63]. The optimum conditions were studied.
Beer's law was obeyed in the concentration range of 0-3.0 μgmL-1. The molar
absorptivity of the method was found to be 8.05×103 Lmol-1cm-1. The method could
be applied to the detection of selenium in tea and mineral water samples with
satisfactory results.
Ensafi and Lemraski described a sensitive and rapid kinetic
spectrophotometric method for the detection of ultra trace amounts of selenium(IV)
[64]. The method was based on the catalytic effect of Se(IV) on the reduction of
sulfonazo by sodium sulfide. The limit of detection was 0.3 ngmL-1 of Se(IV) at 680
nm. The selectivity of the selenium detection was greatly improved using the cation
exchange resin. The method was used for the detection of Se(IV) in a food sample,
natural water and synthetic samples with satisfactory results.
Khajehsharifi et al. reported a kinetic spectrophotometric method for the
simultaneous determination of selenium(IV) and tellurium(IV) [65]. The method was
based upon the catalytic effect of these cations on the reaction of toluidine blue with
sulfide. Partial least squares calibration method was employed for the data
manipulation and analysis. The concentrations were varied between 0.02–0.24 and
0.01–0.08 � �,-1 for Se(IV) and Te(IV) respectively. Cross-validation method was
used to select the optimum number of factors. The root mean square errors of
difference for selenium and tellurium were 1.2 and 1.7 � �,-1 respectively.
197
Application of the method to artificial samples and several mixtures of standard
solutions of Se(IV) and Te(IV) were performed and satisfactory results were obtained.
Liang et al. described a method of kinetic spectrophotometry for the detection
of trace amounts of selenium was established by catalytic kinetics [66]. The
sensitivity of the method was 0.905 μgmL-1. Beer’s law was obeyed in the
concentration range of 0-9.6 μgmL-1. The method was used for the detection of trace
amounts of Se(IV) in Chinese herbal medicine with satisfactory results.
Feng-Shang and Di reported UV spectrophotometric determination of
selenium in black fungus [67]. The method was sensitive and precise with relative
standard deviation (RSD) of 0.054% and the recovery of 97.74%-100.75% and was
applied for the detection of selenium in blank fungus with satisfactory results.
Gudzenko et al. developed a catalytic spectrophotometric determination of
nanogram amounts of selenium(IV) [68]. The method was based on the reduction of
nitrate with iron(II)-EDTA catalyzed by Se(IV) compounds. The reaction proceeded
in several stages and formed iron(III)-EDTA, the nitrosyl complex of iron, nitrous
acid and other products. Nitrous acid entered into the diazotization reaction with
aromatic amine. The resulting diazo compound was coupled with another aromatic
amine to form the azo compound. 4-Nitroaniline was used as the diazo component
and N-diethyl-N′-(1-naphthyl)ethylenediamine was used as the azo component. The
molar absorptivity of the solution of the azo compound was 4.5×104 at 540 nm. The
detection limit of selenium by the proposed method was 0.1 ngmL-1. In the
determination of 0.2 and 2 ngmL-1 selenium, the relative standard deviation was 6 and
2 % respectively.
Zhengjun et al. developed a flow injection catalytic kinetic spectrophotometric
method for rapid determination of trace amounts of selenium [69]. The method was
based on the accelerating effect of Se(IV) on the reaction of EDTA and sodium nitrate
with ammonium iron(II) sulfate hexahydrate in acidic medium. The absorbance
intensity was registered in this reaction solution at 440 nm. The calibration graph was
linear in the range of 5×10+1–2×10+2 and 2×10+2–2×10+3 gmL+�. The detection limit
was 2×10+1 gmL+�. The relative standard deviation was 3.4% for 5×10+� gmL+�
198
selenium(IV) (n=11), 2.7% for 5×10+2 gmL+� selenium(IV) (n = 11). This method was
very simple, rapid and suitable for automatic and continuous analysis. The method
was applied successfully to determination of Se(IV) of seawater samples. Muramoto
et al. reported a novel method for the determination of trace amounts of selenium in
iron and steel has been demonstrated by a HPLC using 2,3-diaminonaphthalene
(DAN) as a derivatizing reagent [70].
Revanasiddappa and Dayananda reported a highly sensitive
spectrophotometric determination of selenium using a reagent leuco malachite green
[71]. The method was based on the reaction of selenium(IV) with potassium iodide in
an acidic condition to liberate iodine, the liberated iodine oxidized leuco malachite
green to malachite green dye. The green coloration was developed in an acetate buffer
(pH 4.2–4.9) on heating in a water bath (40°C). The formed dye exhibited an
absorption maximum at 615 nm. The method obeys Beer’s law over a concentration
range of 0.04–0.4 µgmL+� selenium. The molar absorptivity and Sandell’s sensitivity
of the color system were found to be 1.67×105 Lmol+�cm-1 and 0.5 ngcm+%
respectively. The method was successfully applied to the determination of selenium in
real samples of water, soil, plant material, human hair and cosmetic samples.
Li et al. described a catalytic spectrophotometric method for the determination
of trace amount of Se(IV) in microemulsion medium [72]. The method was based on
the catalytic effect of traces of selenium(IV) on the oxidation of
2�4��-dichlorophenylfluorone by potassium bromate with HNO3 as an activator in the
presence of nonionic microemulsion medium. Under optimum conditions, the
calibration graph was linear in the range of 0.4–��� ,+� of Se(IV) at 480 nm. The
detection limit achieved was 9.86×10+�� � ,+�. Samples were dissolved and the
obtained trace amounts of Se(IV) was separated and enriched by sulphydryl dextrane
gel. The method was applied for the determination of trace selenium with satisfactory
results.
Cherian and Narayana was reported a system for the spectrophotometric
determination of trace amounts of selenium [73]. The proposed method was based on
the oxidation of phenylhydrazine-p-sulphonic acid and the coupling reaction.
Selenium(IV) oxidized phenylhydrazine-p-sulphonic acid into its diazonium salt in an
199
acidic medium. The diazonium salt was then coupled with acetylacetone or ethyl
acetoacetate in an alkaline medium, which formed azo dyes with absorption
maximum at 490 or 470 nm respectively. The method obeyed Beer’s law in the
concentration range of 0.5-20 μgmL-1 of selenium with phenylhydrazine-p-sulphonic
acid-acetylacetone and 1.0-24 μgmL-1 of selenium with phenylhydrazine-p-sulphonic
acid-ethyl acetoacetate couples. The molar absorptivity and Sandell’s sensitivity for
the colored system with phenylhydrazine-p-sulphonic acid-acetylacetone and
phenylhydrazine-p-sulphonic acid-ethyl acetoacetate couples were found to be
1.02×104 Lmol-1cm-1, 7.69×10-3 μgcm-2 and 1.18×104 Lmol-1 cm-1, 6.67×10-3 μgcm-2
respectively.
Mathew and Narayana used azure B as a chromogenic reagent for the
spectrophotometric determination of selenium [74]. The molar absorptivity and
Sandell’s sensitivity of the method were found to be 0.9473×105 Lmol-1cm-1 and
8.33×10-4 μgcm-2 respectively. Beer’s law was obeyed in the range 2.0-10.0 μgmL-1
of selenium.
Kumar et al. described a flow injection spectrophotometric method for the
determination of selenium (IV) in pharmaceutical formulations [75]. The method was
based on the oxidation of 4-aminoantipyrine (4-amino-1,2-dihydro-1,5-dimethyl-2-
phenyl-3H-pyrazole-3-one; 4-AAP) by selenium in presence of acidic medium and
the coupling with N-(naphthalen-1-yl)ethane-1,2-diamine dihydrochloride, which
formed a violet color derivative. Beer's law was obeyed for selenium in the
concentration range 0.05-5.0 µgmL-1 and Sandell's sensitivity was found to be
0.00286 µgcm-2. The reported methods are either not sensitive enough or required
complicated and expensive instruments and are time consuming. The need for a
simple and sensitive spectrophotometric method for the determination of selenium is
therefore clearly recognized.
The aim of the present work is to provide a simple, accurate and sensitive
method for the determination of selenium using toluidine blue and safranine O as new
reagents. The proposed method is well adopted for the determination of selenium in
various environmental and pharmaceutical samples.
200
7.3 APPARATUS
A Secomam Anthelie NUA 022 UV-Visible spectrophotometer with 1 cm
quartz cell was used. A WTW pH 330-pH meter was used.
7.4 REAGENTS AND SOLUTIONS
All chemicals were of analytical reagent grade or chemically pure grade and
double distilled water was used throughout the study. A standard stock solution of
selenium was prepared by dissolving 1.912 g of NaHSeO3 in 1000 mL of water and
standardized by the dithiozone method [42]. Toluidine blue solution (0.02%),
safranine O solution (0.02%), potassium iodide (2%), hydrochloric acid (1M), acetate
buffer solution (pH=4) were used.
7.5 PROCEDURES
7.5.1 Using Toluidine Blue as a Reagent
Aliquots of sample solution containing 1.0–16.0 μgmL-1 of selenium solution
were transferred into a series of 10 mL calibrated flasks. A volume of 1 mL of 2 %
potassium iodide solution was added followed by 1 mL of 1 M hydrochloric acid and
the mixture was gently shaken until the appearance of yellow color, indicating the
liberation of iodine. A 0.5 mL of 0.02 % toluidine blue solution was then added to it
followed by the addition of 2 mL of acetate buffer solution of pH=4 and the reaction
mixture shaken for 2 minutes. The contents were diluted to 10 mL with distilled water
and mixed well. The absorbance of the resulting solutions were measured at 628 nm
against the corresponding reagent blank. A reagent blank was prepared by replacing
the analyte(selenium) solution with distilled water. The absorbance corresponding to
the bleached color which in turn corresponds to the analyte(selenium) concentration
was obtained by subtracting the absorbance of the blank solution from that of test
solution. The amount of the vanadium present in the volume taken was computed
from the calibration graph (Figure VIIA1).
201
7.5.2 Using Safranine O as a Reagent
Aliquots of solution containing 0.8–15.4 μgmL-1 of selenium were transferred
into a series of 10 mL calibrated flasks. A volume of 1 mL of 2 % potassium iodide
solution was added followed by 1 mL of 1M hydrochloric acid and the mixture was
gently shaken until the appearance of yellow color, indicating the liberation of iodine.
A 0.5 mL of 0.02% safranine O and 2 mL of acetate buffer solution of pH=4 were
added to each flask and the reaction mixture was shaken for 2 minutes. The contents
were diluted to 10 mL with distilled water. The absorbance of the resulting solutions
were measured at 532 nm against a reagent blank. A blank solution was prepared by
replacing the selenium solution with distilled water. The absorbance corresponding to
the bleached color, which in turn corresponds to the selenium concentration, was
obtained by subtracting the absorbance of the blank solution from that of the test
solution. The amount of the selenium present in the volume taken was computed from
the calibration graph (Figure VIIA2).
7.5.3 Determination of Selenium in Water
Aliquots (≤5 mL) of water sample containing not more than 15.0 μgmL-1 of
selenium were treated with 0.5 mL of 1M NaOH and 0.5 mL of 0.2M EDTA. The
solutions were mixed and centrifuged to remove the formed precipitate. The
centrifugate was transferred to a 10 mL calibrated flask. They all tested negative. To
these samples a known amount of the selenium was added. An aliquot of the made up
solutions containing selenium was determined directly according to the proposed
method (using toluidine blue or safranine O) and also by the reference method[48].
The results are listed in Table 7A2.
7.5.4 Determination of Selenium in Soil
A known weight (50.0 g) of a soil sludge sample was placed in a 50 mL
beaker and extracted 4 times with a 5 mL portion of concentrated HCl. The extract
was boiled for 10 minutes to convert any Se(VI) present in the soil to Se(IV) cooled
and neutralized (pH =7) with 10% NaOH. A volume of 5 mL of 5 % EDTA solution
was added and the contents were made up to 25 mL with water. An aliquot (≤5 mL)
of the made up solution containing selenium was determined directly according to the
202
proposed method (using toluidine blue or safranine O) and also by the reference
method[48]. The results are listed in Table 7A2.
7.5.5 Determination of Selenium in Pharmaceutical Samples
A volume of 10 mL of each Fourts B (Fourts India Laboratories Private Ltd.,
Kelambakkam-603 103, Tamil Nadu, India) and Homoxid (Angle French drugs,
India) samples were treated with 10 mL of concentrated HNO3 and the mixture was
then evaporated to dryness. The residue was leached with 5 mL of 0.5 M H2SO4. The
solution was diluted to a known volume with water after neutralizing with dilute
ammonia. An aliquot of the made up solution was analysed for selenium according to
the general procedure described earlier. The results are listed in Table 7A2.
7.6 RESULTS AND DISCUSSION
7.6.1 Absorption Spectra
This method involves the liberation of iodine by the reaction of selenium with
potassium iodide in an acidic medium. The liberated iodine bleaches the blue color of
toluidine blue and absorbance of the solution is measured at 628 nm. This decrease in
absorbance is directly proportional to the selenium concentration. At the same time
the liberated iodine bleaches the pinkish red color of safranine O and the absorbance
of the solution is measured at 532 nm. This decrease in absorbance is directly
proportional to the selenium concentration. The absorption spectra of colored species
of toluidine blue and safranine O is presented in Figure VIIA3 and reaction system is
presented in Scheme VII.
7.6.2 Effect of Iodide Concentration and Acidity
The effect of iodide concentration and acidity on the decolorization is studied
with 5 μgmL-1 of selenium solution. The oxidation of iodide to iodine is effective in
the pH range 1.0 to 1.5, which could be maintained by adding 1 mL of 1 M HCl in a
final volume of 10 mL. The liberation of iodine from KI in an acidic medium is
quantitative. The appearance of yellow color indicates the liberation of iodine.
Although any excess of iodide in the solution did not interfere. It is found that 1 mL of
2 % KI and 1 mL of 1 M HCl were sufficient for the liberation of iodine from iodide
203
by selenium and 0.5 mL of 0.02 % toluidine blue or 0.02 % safranine O is used for
subsequent decolorization.
Constant and maximum absorbance values are obtained in the pH=4±0.2.
Hence the pH of the reaction system was maintained at 4±0.2 throughout the study.
This could be achieved by the addition of 2 mL of 1 M sodium acetate solution in a
total volume of 10 mL. In the case of toluidine blue method, the bleached reaction
system is found to be stable for more than 6 hours and also in safranine O reagent
case, the bleached reaction system is found to be stable for 4 hours.
7.6.3 Analytical Data
7.6.3.1 Using toluidine blue as a reagent
The adherence to Beer’s law is studied by measuring the absorbance values of
solutions varying selenium concentration. A straight line graph is obtained by
plotting absorbance against concentration of selenium. Beer’s law obeyed in the
range of 1.0–16.0 μgmL–1 of selenium (Figure VIIA1). The molar absorptivity and
Sandell’s sensitivity of the system is found to be 1.240×104 Lmol-1cm-1 and 6.37×10-3
μgcm-2 respectively. Correlation coefficient (n=10) and slope of the calibration curve
are 0.9992 and 0.150 respectively. The detection limit (DL=3.3 σ/s) and quantitation
limit (QL=10 σ/s) [where σ is the standard deviation of the reagent blank (n=5) and
s is the slope of the calibration curve] of selenium determination are found to be 0.220
μgmL-1 and 0.670 μgmL-1 respectively. Adherence to Beer’s law graph for the
determination of selenium using toluidine blue is presented in Figure VIIA1.
7.6.3.2 Using safranine O as a reagent
The adherence to Beer’s law is studied by measuring the absorbance values of
solutions varying selenium concentration. A straight line graph is obtained by
plotting absorbance against concentration of vanadium. Beer’s law is obeyed in the
range of 0.8 – 15.4 μgmL-1 of selenium (Figure VIIA2). The molar absorptivity and
Sandell’s sensitivity of the system is found to be 1.190×104 Lmol-1cm-1, 6.63×10-3
μgcm-2 respectively. Correlation coefficient (n=10) and slope of the calibration curve
are 0.9995 and 0.154 respectively. The detection limit (DL=3.3 σ/s) and quantitation
limit (QL=10 σ/s) [where σ is the standard deviation of the reagent blank (n=5) and
204
s is the slope of the calibration- curve] for selenium determination are found to be
0.214 μgmL-1 and 0.649 μgmL-1 respectively. Adherence to Beer’s law graph for the
determination of selenium using safranine O is presented in Figure VIIA2.
7.6.4 Effect of Diverse Ions
The effect of various ions at microgram levels on the determination of
selenium is examined. The tolerance limits of interfering species are established at
those concentrations that do not cause more than �5%6������������������������!
��������"&'(� �� �,-1. Ions such as Ni2+, Cu2+, Al3+, Co2+, V5+, Fe3+, sulfate are
interfered. However, the tolerance level of some of these ions may be increased by the
addition of 1 mL of 1 % EDTA solution and the interference of Fe3+ was masked
using sodium fluoride solution. The tolerance limits of various foreign ions are given
in Table 7A1.
7.7 APPLICATIONS
The developed method is applied to the quantitative determination of selenium
in various environmental and pharmaceutical samples. The results of are presented in
Table 7A2 and the analysis of the above samples are compared with those from a
reference method [48]. The precision and accuracy of the proposed method is
evaluated by replicate analysis of samples containing selenium at two different
concentrations.
7.8 CONCLUSIONS1. The reagents provide a facile, rapid and accurate method for the spectrophotometric
determination of selenium.
2. The reagents have an advantage of high sensitivity and selectivity.
3. The method needs neither heating for the complete color development nor
extraction into any organic phase.
4. The accuracy of the method is comparable with most methods reported in the
literature.
5. The proposed method is used for the determination of traces of selenium in
various environmental and pharmaceutical samples. A comparison of the method
reported is made with earlier methods and is given in Table 7A3.
205
FIGURE VIIA1ADHERENCE TO BEER’S LAW FOR THE DETERMINATION OF SELENIUM
TOLUIDINE BLUE AS REAGENT
Concentration of selenium (µgmL-1)
0 2 4 6 8 10 12 14 16 18 20
Abs
orba
nce
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
FIGURE VIIA2
ADHERENCE TO BEER’S LAW FOR THE DETERMINATION OF SELENIUM
USING SAFRANINE O AS A REAGENT
Concentration of selenium (µgmL-1)
0 2 4 6 8 10 12 14 16 18
Abs
orba
nce
0.0
0.2
0.4
0.6
0.8
1.0
1.2
206
FIGURE VIIA3ABSORPTION SPECTRA OF COLORED SPECIES OF TOLUIDINE BLUE (A)
AND SAFRANINE O (B)
W avelength (nm)
200 300 400 500 600 700 800 900
Abs
orba
nce
0 .0
0.2
0.4
0.6
0.8
1.0
1.2
a
b
SCHEME VII
H2SeO3 + 4I- + 4H+ Se + 2I2 + 3 H20
N
S+
CH3
NH2(CH3)2N
NH
S
CH3
NH2(CH3)2N
I2 , H+
Toluidine Blue (Colored) Toluidine Blue (Colorless)
N
N+
CH3
NH2NH2
CH3 NH
N
CH3
NH2NH2
CH3 I2 , H+
Safranine O (Colored) Safranine O (Colorless)
207
TABLE 7A1
EFFECT OF DIVERSE IONS ON THE DETERMINATION OF SELENIUM (5 μgmL-1)
Foreign ionsTolerance limit (μgmL-1) Foreign ions
Tolerance limit (μgmL-1)
Ni2+*
Cu2+ *
Cd2+
Ba2+
Fe3+*
Bi3+
Al3+*
Ca2+
75
50
100
200
75
200
50
200
Co2+*
V5+*
Zn2+
Tartarate
Oxalate
PO43-
Sulfate*
Glucose
75
75
200
500
500
250
50
200
*Masked by secondary masking agents.
208
TABLE 7A2DETERMINATION OF SELENIUM IN VARIOUS ENVIRONMENTAL AND
PHARMACEUTICAL SAMPLES USING TOLUIDINE BLUE AND SAFRANINE
O AS REAGENTS
Toluidine blue Safranine O Samples Se(IV) Se(IV) Recovery RSD Se(IV) Recovery RSD
added* found* (%) (%) found* (%) (%)a Tap Water 4.00 3.96 99.00 0.65 3.98 99.50 0.51
Samples 8.00 7.92 99.00 0.75 7.95 99.38 0.88a Rain Water 4.00 3.98 99.50 2.50 3.97 99.25 1.81
Samples 8.00 7.96 99.50 0.85 7.98 99.75 0.65a Industrial Water 4.00 3.97 99.25 1.51 3.95 98.75 0.58
Samples 8.00 7.95 99.37 0.76 7.97 99.63 1.35 (Collected from the industrial
zone of Mangalore city)
Soil Samples ___ 1.35 ___ 0.74 1.32 ___ 2.03
4.00 5.36 100.25 1.34 5.28 99.00 1.37
8.00 9.31 99.50 0.92 9.27 99.37 0.97abFourts B 4.00 3.99 99.75 0.40 3.96 99.00 0.76
8.00 7.95 99.37 0.75 7.92 99.00 1.04acHomoxid 4.00 3.96 99.00 1.21 3.94 98.50 1.76
8.00 7.91 98.87 1.11 7.93 99.13 0.75
7�� �,-1
a. Selenium was not detected in ground water, tap water, industrial water and
pharmaceutical samples.
b. Fourts B (Fourts India Laboratories Private Ltd., Kelambakkam-603 103, Tamil
Nadu, India) Composition –thiamine mononitrate-10 mg; riboflavin-10 mg;
pyridoxine hydrochloride-3mg; vitamin C-75 mg; zinc sulphate-55 mg; selenium-
100 µg; folic acid-1mg; niacinamide-50µg; chromium-200µg; L-cysteine HCl-
25mg; glycine-25mg, glutamic acid-25mg; vanadium-100µg;
c. Homoxid (Angle-French drugs, India) Composition – pyridoxine HCl-10mg; folic
acid-1mg; cyanocobalamin-0.4mg; vit c-150mg; β-carotene-10mg; selenium-70µg;
209
TABLE 7A3COMPARISON OF THE METHOD REPORTED IS MADE WITH EARLIER
METHODS
ε = Molar absorptivity, ss = Sandell’s sensitivity
Reagent Method Beer’s law"� �,-1)
ε (Lmol-1cm-1)��"� ��-2)
λmax Ref. No.
5,5-dimethyl-1,3-cyclohexanedione
Spectrophotometry upto 30 ε = 4.00×103
ε = 3.77×103313300
48
Variamine Blue Spectrophotometry 2.0-20 ε = 2.60×104
ss = 3.0×10-3546 58
Starch Spectrophotometry 2.0-�%� ε = 1.40×104
ss = 5.45×10-3570 61
Leuco malachite green
Spectrophotometry 0.04-0.4 ε = 1.67×105
ss = 0.50 ngcm-2615 71
Ethyl acetoacetate Spectrophotometry 1.0-24 ε = 1.18×104
ss = 6.67×10-3470 73
Acetylacetone Spectrophotometry 0.5-20 ε = 1.02×104
ss = 7.69×10-3490 73
Azure B Spectrophotometry 2.0-10 ε = 0.947×105
ss = 8.33×10-4644 74
Proposed Method
Toluidine blue
Safranine O
Spectrophotometry
Spectrophotometry
1.0-16.0
0.8-15.4
ε = 1.240×104
ss = 6.37×10-3
ε = 1.190×104
ss = 6.630×10-3
628
532
210
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