ANALYTICAL SCIENCES JUNE 1998, VOL.

14 479
1998 © The Japan Society for Analytical Chemistry

Reviews

Speciation of Selenium Compounds

Krystyna Pyrzyńska

Department of Chemistry, University of Warsaw, Pasteura 1, 02–093 Warsaw, Poland

The toxicity and essential nature of selenium in the environment depend on its concentration and on the chemical forms in
which it is present. Dissolved inorganic selenium can be found in natural waters as selenides (Se–II), colloidal elemental
selenium (Se0), selenite (SeIV) as well as selenate (SeVI). Organic forms of selenium that may be found in organisms, air
or in the aqueous environment are volatile methylselenides, trimethylselenonium ion and selenoamino acids. This review
article intends to show some general ideas in speciation analysis of selenium and to highlight some important procedures.

Keywords Selenium, speciation, environmental analysis

1 Introduction 479 5 Selenoamino Acids 481

2 Determination of Total Selenium 480 6 Volatile Alkylselenides 482
3 Selenite and Selanate 480 7 Conclusion 482
4 Trimethylselenonium Ion 481 8 References 482

teins and their derivatives. Inorganic selenium com-

1 Introduction pounds can be transformed into volatile compounds
such as dimethylselenide (DMSe) and dimethyldi-
In recent years, there has been increasing interest in selenide (DMDSe) through microbial action of fungi,
the trace determination of selenium. This element has plants and animals. The trimethylselenonium ion
been recognized as an essential nutrient for humans (TMSe+), the major product of selenium metabolism,
based on its presence in the enzyme glutathione peroxi- leaves the body of humans in urine. The biomethyla-
dase, which affords cells protection against oxidative tion processes are considered to be detoxification steps,
damage.1,2 Selenium compounds also catalyze the reac- because DMSe and TMSe+ are less toxic than other
tions of intermediate metabolism and inhibit the toxic selenium compounds.
effects of heavy metals such as arsenic, cadmium, mer- The development of reliable techniques to study the
cury and tin. 3 The recommended daily dietary speciation of selenium in environmental and biological
allowances of selenium for women and men are 55 mg samples is necessary to understand the biochemical
and 70 mg per body, respectively.4 On the other hand cycle, mobility and uptake of selenium, as well as its
at higher concentrations selenium is toxic.1 The maxi- toxicity. Many problems in selenium speciation analy-
mum recommended5 selenium concentration in drink- sis are associated with the low concentration of each
ing water is 10 +g l –1. Apart from natural sources species to be determined. Significant errors may also
(mainly metal-sulfur minerals), selenium compounds arise by losses of volatile selenium compounds, insta-
are widely spread throughout the environment from the bility of its chemical species, contamination, etc.
combustion of fossil fuels, and uses in the glass and A number of authors have presented various analyti-
electronics industries and in agriculture. cal procedures for speciation of selenium. Compared
The toxicity and essential nature of Se depend on its with the extensive investigations on total selenium or
chemical form. Selenium naturally exists in several selenite and selenate determination, very little work has
oxidation states in inorganic and organic forms. The been carried out on organic selenium compounds. We
inorganic selenium species most frequently found in have neither the intention of covering all published
water and soils are selenite (SeO 3 2– ) and selenate methods, nor of criticizing specific papers. We would
(SeO42–). Several organoselenium compounds have rather discuss some general ideas and highlight some
been identified in biological samples (animals, plants, important procedures.
microorganisms) with direct Se–C bonds including
methylated compounds, selenoamino acids, selenopro-
480 ANALYTICAL SCIENCES JUNE 1998, VOL. 14

2 Determination of Total Selenium

The first step in speciation analysis of selenium is

determination of the total metal content, usually carried
out after mineralization of organic matrix. Several ana-
lytical techniques have been applied for this purpose.
The most important are fluorometry, polarography and
voltammetry, hydride generation and graphite furnace
atomic absorption spectrometry, mass spectrometry and
Fig. 1 Flow-injection system for speciation of inorganic sele-
neutron activation analysis. These methods have been nium by HG-AAS (adopted from ref.10).
recently reviewed.6–9 Selenium reactivity and bioavail-
ability depend not only on its total amount. Additional
knowledge of the chemical forms and oxidation states
in which this element can exist is needed. tion.12–14 The DPCSV method is based on the deposi-
Proposed speciation procedures determine the follow- tion of a HgSe film on a hanging drop electrode, fol-
ing main groups of Se compounds: lowed by the dissolution of this film by reduction of
• inorganic selenium species such as SeIV and SeVI HgII. The presence of dissolved organic matter, mostly
• trimethylselenonium ion as humic substances, strongly disturbs the measurement
• selenoamino acids and standard addition procedures should be used.13
• volatile methylated forms such as DMSe and DMDSe Electrochemical methods are specific for Se IV. Their
application for inorganic selenium species needs com-
bination with chemical treatments. SeIV is measured
3 Selenite and Selanate directly in one subsample, and another subsample is
treated with HCl to reduce SeVI to SeIV. The concen-
Selenite and selenate are the most mobile and biogeo- tration of selenate is calculated as the difference.
chemically important forms of selenium. Most of the Obviously, any error which occurs in either measure-
studies published so far on selenium speciation deal ment for SeIV or total inorganic selenium will automati-
with these two species. The ratio of SeIV to SeVI in nat- cally affect the result for SeVI. As in the hydride gener-
ural waters depends on the presence of acids (pH), ation technique, this is an inherent disadvantage with
complexing agents, dissolved gases (especially oxygen) this method.
and suspended matter. Fluorometric methods are based on the detection of
Hydride generation coupled to AAS or ICP detectors, the fluorescent complex formed between Se IV and
is the technique most commonly used for speciation of mainly 2,3-diaminonaphtalene or its derivatives.15,16
inorganic selenium. Se(IV) is selectively reduced to Only SeIV can be determined by this method, so specia-
volatile SeH2 usually by sodium tetrahydroborate in tion is limited by the ability to transform each species
hydrochloric acid medium. Since this reaction is spe- to Se IV . Recently, automatic spectrofluorometric
cific for Se IV, this technique can be applied for the method for the simultaneous determination of SeIV and
determination of total inorganic selenium (the sum of SeVI using flow-injection techniques has been devel-
SeIV and SeVI) after quantitative reduction to selenite. oped.17 The method is based on the selective oxidation
The content of Se VI is obtained as the difference of the non-fluorescent reagent 2-( _ -pyridyl)thio-
between two determinations. Hydride generation of quinoldiamide) by SeIV to give an intensely fluorescent
selenium can be carried out by batch process, flow- oxidation product. SeVI is reduced on line in a coil
injection analysis or by a sampling continuous flow installed in a photo-reactor and determined from the
system. The flow-injection system offers advantages of difference between two fluorescences values.
full automation, quick analysis and minimum sample Graphite furnace atomic absorption spectrometry may
and reagent consumption. Since selenium may be lost be applied to the determination of SeIV and SeVI provid-
during the prereduction step owing to the formation of ing that chemical reactions are carried out prior to
volatile selenium chloride, an on-line reduction in a detection, such as complex formation followed by sol-
closed system at 140˚C, shown in Fig. 1, is proposed.10 vent extraction18,19, separation of species on solid sor-
This process can be also performed using microwave bents20–22 or selective coprecipitation.23 In an effort to
heating.11 The generated selenium hydride is enriched improve the thermal stability of selenium in a graphite
into a liquid nitrogen cold trap and then swept into a furnace, various modifiers have been proposed. 24–26
flame or electrically heated quartz tube or into a Chemical modification was found to be affected by the
graphite furnace for detection. valence state of selenium, which is of great importance
Among various proposed electroanalytical tech- in routine analysis.26,27
niques, differential pulse polarography (DPP) and dif- High performance liquid chromatography (HPLC)
ferential pulse cathodic stripping voltammetry coupled with selenium-specific detection, such as AAS,
(DPCSV) are the most applied for selenium specia- ICP-AES, ICP-MS, have been used to great advantage
ANALYTICAL SCIENCES JUNE 1998, VOL. 14 481

in inorganic selenium speciation analysis. Selenite and

selenate are commonly separated by anion-exchange
chromatography28–31, although ion-pair reverse-phase
chromatography can also be employed.32,33 The com-
parison of three selenium-specific devices as chromato-
graphic detectors showed that the absolute detection
limit (for 100 +l injection onto the column) was 10 ng,
1 ng and 0.1 ng of Se for FAAS, GFAAS and ICP-MS
detectors, respectively.29 The last system showed also
the greatest linear calibration range of 0.01 – 10 mg l–1.

4 Trimethylselenonium Ion

Trimethylselenonium ion is a minor metabolite of

selenium which is mainly present in urine and water Fig. 2 Speciation scheme for selenium analysis.
samples. Its content is useful in predicting the excess
Se intake and the detoxification mechanism in the liv-
ing organism. However, the chemical forms of seleni-
um in urine have not yet been completely character- or electrochemical methods; (2) after oxidation by UV
ized. irradiation or wet acid digestion_the sum of Se–II and
Some of the methods used for determination of SeIV, and (3) after mineralization of organic matrix fol-
TMSe+ are based on ion-exchange chromatography and lowed by reduction of selenate to selenite with
fractional quantification. Because of its cationic hydrochloric acid_all selenium species. The difference
nature, TMSe+ is preconcentrated and separated, usual- between total selenium and inorganic species is attrib-
ly from inorganic selenium species, on the cation- uted to organic selenium compounds. Usually, a pre-
exchange columns.18,31,34 concentration step (solvent extraction, coprecipitation,
The high resolution obtained by HPLC permits a sorption on solid sorbents) is required to achieve a suf-
clear separation of TMSe+ from inorganic selenium and ficient concentration level for detection. The proposed
also from other organoselenium compounds. 28,29,32,35 analytical procedures differ mainly in the way in which
Reversed-phase partitioning and ion exchange chro- selenium species are converted into SeIV and in the
matography have been applied. The stationary phase is methods applied for preconcentration.
usually silica, bonded or coated. Phosphate or citrate The separation of the dissolved organic selenide frac-
buffer is applied as mobile phase which contains a tion can be achieved by using liquid chromatography at
polar organic solvent (mainly methanol) to get signal atmospheric pressure.31,34,41–43 Organoselenium species
enhancement and good chromatographic resolution. (other than TMSe +) adsorbed on Amberlite XAD-2
The TMSe+ level was also calculated indirectly by resin at pH 8 were classified as neutral and basic forms,
forming the volatile compound dimethyl selenide after while those absorbed at pH 3 were classified as
thermal decomposition of trimethylselenonium in high- acidic.31 As the acidic forms dominated in lake water
ly alkaline solution. 31 The generated DMSe was samples, it was concluded that most of these com-
adsorbed in concentrated nitric acid and analyzed by pounds were selenium-containing humic substances.
mass spectrometry. Nevertheless, the most widely used method for deter-
mination of selenoamino acids is HPLC and this is
applied to many matrices.44–47 In all these studies, the
5 Selenoamino Acids compatibility of the mobile phase with the detector
strongly affects the detection limit. For instance ther-
Among naturally occurring selenium species, mochemical THG-AAS requires a high methanol-con-
selenoamino acids play an essential role. With regard taining eluent44, whereas ICP-MS sensitivity is affected
to humans, inorganic compounds would be less by saline solutions45 and GF-AAS needs the use of a
absorbed than selenomethionine and this latter form matrix modifier that enhances selenium detection. 46
would serve for selenium storage in proteins for its Thus, the conditions for chromatographic separation
eventual use in selenocysteine synthesis and its incor- of selenoamino acids have to be closely adapted to
poration into selenium-specific enzymes.36,37 the detector. Using the HPLC method, several
Many procedures involving selenium speciation selenoamino acids and related selenium compounds
determine selenoamino acids in the fraction named have been identified in biological tissues or protein
organic selenium compounds.13,15,38–40 The scheme, as fractions. Tyson et al.45 analyzed by the HPLC-ICP-
is shown in Fig. 2, analyses three separate sample MS method several vegetables (garlic, onion, broccoli),
aliquots: (1) with no further chemical treatment_deter- which were grown under standard nutrition conditions
mination of SeIV using fluorometry, HG-AAS, HG-ICP with addition of sodium selenate. This group of veg-
482 ANALYTICAL SCIENCES JUNE 1998, VOL. 14

etables tend to take up inorganic selenium, followed by

conversion to various organic forms. Five selenoamino 7 Conclusion
acids were identified in the extracts from these samples
and some unknown peaks were also observed (Fig. 3). In recent years, the biochemistry of selenium species
Also, capillary electrophoresis provides the opportu- has been the object of increasing attention due to the
nity for rapid and simple analysis of selenoamino acids importance of this element as both an essential and a
together with high efficiency. 49–52 This method led toxic substance. However, the accurate speciation of
to low detection limits, usually in the +g l–1 range with selenium is still a major challenge for analytical
a small sample volume of only a few nanoliters. chemists. While a majority of methods for the determi-
Nevertheless, the lack of selectivity and sensitivity of nation of selenium in environmental and biological
the UV detector is a limit to its application to more spe- samples give the total selenium content, only a few pro-
cific and sensitive complex matrices. The use of a cedures are capable of distinguishing the different
more specific detection method such as ICP-MS and chemical forms of this metal. This methods which sep-
also the study of preconcentration procedures closely arate the individual species, followed by their direct
adopted to capillary electrophoresis analysis, appears to separation, are preferred due to the need for only mini-
be a necessary step which would make this technique mal sample pretreatment. The last factor is particularly
more attractive for environmental and biological analy- important because prolonged sample manipulation may
sis. affect the distribution of selenium species significantly.
The most efficient way for the quality control of ana-
lytical results is through the analysis of certified refer-
6 Volatile Alkylselenides ence materials. Although several certified reference
materials for Se with different matrices are available,
Volatile methylated selenium species such as DMSe they are only certified for total selenium content.7 The
and DMDSe are produced from inorganic selenium Measurements and Testing Programme (formerly BCR)
through a biomethylation process by various organisms. has initiated a project to prepare certified reference
Rats feed with selenite and selenate exhaled a volatile material of inorganic species (selenite and selenate) in
compound identified as DMSe.53 This compound and aqueous solution.59 The interlaboratory studies on sta-
also DMDSe have been observed emanating from soils, bility of organoselenium species have already started60
biologically active lake sediments and sewage sludge.8 with the purpose to prepare further new reference mate-
Volatile alkylselenides are sampled from air or water rials in biological tissues, such as fish.
and trapped either on a solid adsorbent or in a liquid
nitrogen trap, followed by their thermal desorption This study was supported by grant from the Nation Committee
for Science Research, Project nr 3 TO9A 016 10.
prior to analysis. DMSe and DMDSe are sufficiently
volatile to be separated directly by gas chromatography
using packed54,55 or capillary columns.54,56,57 The chro-
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