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ANALYSIS OF SELENIUM

Sager, M. Landwirtschaftlich-Chemische Bundesanstalt Wien, Wien, Austria

- 1. DECOMPOSITION METHODS FOR THE DETERMINATION O F TOTAL CONTENTS OJSELENIUM

Most methods for separation and final determination start from a Se (IV) containing sample solution. Suitable decomposition procedures should yield such sample solutions from the various materials encountered, TO check the decomposition procedure, the following methods can be applied: Regain of inorganic selenium added to the sample solution just before final determination means that the determination from this solution is possible as such. The regain of inorganic selenium before starting the decomposition procedure means that this has not been volatilized; organically bound Se may behave in a different way. Labelling with 75-Se tracer simplifies the detection of loss and regain, whatever Se- containing compound has been formed. The behaviour of organically- bound Se has been examined by addition of selenocysteine, selenomethionine, and triniethylselenonium chloride, which are known to occur in selenium metabolism [1,2], in case of mineral oil, dilauryl selenide has been added as a test substance [3]. The most reliable, but also most laborious way appears to control the method with metabolized tracer-selenium Radioactive selenium compounds are injected or ingested by the test animals, which is metabolized in 1-2 days. The labelled tissues and body- fluids can be investigated as well as by radioactive counting as well as by wet- chemical methods (1,2,4,5]. Reagent blanks have been only explicitely given for chloric acid [ 6 ] ; sometimes, sulfuric acid may also contain some selenium Blanks of magnesium nitrate are usually sufficiently low. Running blank samples in parallel, however, is recommended.

1.1. SAMPLES FROM ORGANIC- BiOLOGICAL ORIGIN

Organic compounds of the matrix very often interfere with common analytical methods, as can be easily shown by addition of selenite to incompletely mineralized samples: they block separation columns, make foam in course of hydride evolvation, make smoke in the graphite furnace for AAS, alter electrode surfaces etc. Therefore, it is recommendable to mineralize the sample as complete as possible without losses As selenate i s not detected by many determination methods, the selenate which may have been formed by excess of oxidants, has to be reduced to selenite again. In case of drying of plant and animal tissues as well as body fluids up to 120" or with microwaves, no loss of selenium was observed [2] Labelled selenium fed to rats and metabolized in blood, brain, lungs and muscles, was regained at more than 95% upon drying. Similarly, no losses were found for freeze- drying [7]. Losses, however, may occur during drying of sewage sludges (author's experience) because of reducing conditions.

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1 .1 .1 DRY- ASHING PROCEDURES

Organic material is oxidized by the oxygen of air during heating in a muffle furnace. High sample weights lower the needs for sample homogeneity, which is not so easy to achieve for foods and plants. Addition of magnesium nitrate, (e.g. as 10% solution), careful drying of the mixture, and sufficient ventilation are essential to yield white ash and to keep the sample alkaline [5,8]. After charring at SO0-55O0, the ash is dissolved in l i l - HCI and heated half an hour on the water bath to reduce possibly formed selenate [8,9,10]. Quartz or glass beakers are preferable; for Pt- dishes, losses are reported to occur [ 131. From non- alkaline dry-ashing, however, Se is volatile as oxide or halogenide [12,13]. The reliable destruction of organic matter yields an excellent sample solution for hydride-AAS determination, but it is difficult to employ graphite- furnace- AAS or atomic emission methods. In a stream of air, selenium is quantitatively volatilized from biological material at 600-700", and can be caught by soda lime [ 121.

1.1.2 DECOMPOSITION BY USE OF OXIDIZING ACIDS

I 1 2 1 Closed vessels Decomposition of organic samples with specially purified nitric acid at high pressure in vessels made from PTFE or glassy carbon [ 14,15,16] enables the complete destruction of micro- amounts of sdample [6] with minimum amounts of reagent and thus extremely low blanks The mineralization of metabolized Se has been controlled by gel electrophoresis [ 171 As the organic matrix yields gaseous pioducts during its reaction with nitric acid, the sample weight is, however, limited The resulting solution is saturated with nitrous acid, which interferes with some separation and determination methods of Se

1.1.2.2 Open vessels After addition of the oxidant (usually nitric acid), it is better not to start with vigorous heating, but to wait some time, and to increase temperature slowly. Charring on the sand bath without temperature control leads to volatilization losses. The use of a reflux condenser, or at least long-necked vessels, is recommended. HNO, alone is not sufficient for complete destruction; it has been used for dissolution of blood and food prior to graphite- furnace- AAS [ 181 or prior to GC- determination with 5 - nitropiazselenol [ 17,191. The oxidation power of nitric acid can be increased by addition of H,O, (dropwise, because of gas evolvation) [5,20,2 I], and/or temperature increase by refluxing in admixture with perchloric and/or sulfuric acid, at least in a Kjeldahl- flask [ 1,3,4,1 1,22-381. Refluxing with nitric/sulfuric acid could even decompose coal samples without losses [35]. Gel chromato- graphy of the decomposition solution containing labelled metabolites of selenium showed, that Se was completely mineralized from animal tissue within 1 5 min under reflux with HNO,/H,SO,, but a lot of other non- destructed material may interfere with further steps of analysis [39]. After sufficient refluxing to destruct most of the organics, the rest of nitric acid can be fumed off with perchloric or sulfuric acid; selenium is converted into selenate, and thus not volatile any more [4,5,6,20,22,23,26,28,30,32]. Finally, the ashing temperature can be raised to a maximum of 210" [4,23,30]. This is important for the decomposition of fat and

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other resistant compounds [4,28] Selenium itself is hardly present in the lipoid phase [4,5], but in case of high fat contents, it is necessary to add a 10-15- fold excess of acid (by weight), to avoid reducing conditions [27] Tiimethylselenonium, the main metabolite in urine, is not volatile, but it needs about 20 min i n boiling HNO,/HCIO, = 1 1 to convert it to inorganic selenium ( 1 ) Decomposition with aqua regia, as usual for inorganic samples (including soils), has not been reported biological matrices so far If a mixture containing chloric acid IS used, the resulting solution can be even used for the determination of iodine [6] Open flames nearby have to be avoided, because of explosions If only H,O, is used as oxidant, little addition of acid (perchloric, sulfuric) is also necessary [40,41] After warming, the reaction is exotherm, and not sufficient in any case, it has been used for the mineralization of serum and animal tissues prior to coprecipitation with tellurium, which is interfered by nitric acid [42] Addition of K,Cr,O, [43] as well as catalysts like V 0, [ 3 I ] or Na-Moo, [29,44] to the acid decomposition mixture enables to lower the temperature for destruction, but the resulting sample solution is not well suitable for the analysis of other trace elements, not every selenium- organic compound is mineralized by chromate Vanadium acts as a visual indicator for the end of the decomposition, by changing its coloui from brown to green [311

1.2 DECOMPOSlTlON OF INORGANIC SAMPLES

During the destruction procedure of biological materials i t is above all iiiiportaiit to mineralize selenium from its organic cleavages, and to oxidize a large amount of sample weight without losses and import of blanks In case of inorganic samples, however, its volatility as the fluoride, chloride, and bromide causes troubles, fluorination of silicates I S

not recommendable Under reducing conditions, lihe during the dissolution of sulfides 01

metals, selenium can be lost as H,Se or left undissolved as elementary selenium

I .2.1 DECOMPOSITION I N CLOSED VESSELS

For rocks, sediments and soils, good experience has been made with HNO,/HF = 7 1 (author) Slags and dusts with matrix Cu 01 Pb have been decoinposed wlth HCI HNO, HCIO, = 3 3 1 at 110-150"[43], i n cast: of furnace dust HF HNO, = I I was used [45] For soils, regain of Se was complete after decomposition with HNO, only at 140" 111 the pressure bomb [46]

1.2.2 DECOMPOSITION IN OPEN VESSELS

Soils can be quantitatively extracted for selenium without losses with aqua regia. if reflux condensers are applied [46] Without reflux condensers, it is recommended to worh at the boiling water bath Zinc ore could be dissolved with HNO,/HCI = 3 I on the warer bath, after evaporation of HCI, HNO, could be fumed off with HCIO, [47] Cu and Cu- alloys can be dissolved in l i l - HNO,, transferring also selenide and telluride into quadrivalent oxidational status HCl

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must be avoided, since it produces gaseous hydrogen selenide and selenium chlorides [48]. After evaporation on the boiling water bath, the sample can be fumed off with HCIO, [49]. Steel, however, is resistant towards pure nitric acid; a mixture containing HCI:HNO,: H,O = l:l:2 was used, which can also attack Se(0). Prior to the final determination, HCI and HNO, can be replaced by HCIO, /H,PO, by short heating [50]. Geological material has been decomposed in the classical way with NaOH/Na,O, at 600" after irradiation and addition of a carrier, with subsequent dissolution in hot water and filtration [ 10,591. Acid decompositions with activated material (rocks, soils) were done in open vessels after addition of inactive selenium carrier with HNO, /HCIO,IHF [52], with H2S0,/H20, [46], or with H,SO,/HNO, at 1 50°(air condenser)[53].

1.2.3 DISTILLATION AS BROMIDE

From acid solution, selenium can be distilled as SeBr, and adsorbed in cold water. HNO, interferes, and has to be evaporated or decomposed beforehand. The distillation has a low sample throughput, but it simultaneously offers a highly selective separation from nearly all elements, and it results in a solution of Se(IV), which is wanted for most final determinations. Soils containing selenite, only need addition of HBr [54]. From meteorites, SeBr, was distilled after reflux with sulfuric acid, SeBr, was distilled in a N,- stream with addition of HC1:HBr = 3.1; it is only accompanied by As, Sb, Sn, Re, and Hg [51] . Use of NH,Br instead of HBr avoids the carry-over of acid. Se (IV) and Se(V1) are volatilized from rocks and sediments by heating in admixture with ammonium bromide and phosphoric acid at 250", in a stream of air, and can be retained in weak acid solution. Selenide and elementary selenium do not react and can thus be discriminated. If KJO, has been added, all inorganic forms are distilled together [55,56,57].

1.3 BURNING AND VOLATILIZATION IN A STREAM OF OXYGEN

Heating in a stream of oxygen can volatilize selenium from several inorganic and organic samples as SeO?, which is subsequently condensed on a cool finger, or adsorbed on a proper substrate. From the condenser, selenium can be dissolved with dilute nitric or acetic acids. If selenium is adsorbed on a filter cartridge made of quartz wool/ soda lime/silver wool, it can be directly counted after irradiation of the sample [9,10,12]. Beneath decomposition, a separation from many matrix elements as well as significant enrichment are achieved; the sample output, however, is moderate. Whereas at 1 1 OO", the volatilization of selenium is quantitative, it is only marginal in a mixture of N2 and H2

Ores, metals, minerals, rocks, soils, coals, ashes etc. are weighed into a sample boat and transferred into a quartz- tube, which can be put into a stream of oxygen and heated from outside [43,58]. For a big apparatus, a water-cooled condenser is sufficient; micro- amounts from pure substances were collected in a trap of liquid nitrogen [59]. In case of organic samples, volatilization of unreacted matrix compounds including selenium- organics can be faster than the reaction of burning. This can be prevented by a platinum gauze which is additionally fed with oxygen for catalytic oxidation [60]. For the combustion of micro- amounts of samples, a special apparatus, named Trace-0-Mat, has been constructed, where the sample is ignited electrically in a stream of oxygen. The products of burning are depleted upon a cool finger filled with liquid nitrogen.

~ 3 1 .

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Addition of cellulose facilitates the burning of inorganic matrices, addition of silica retains accompanying elements (e.g. Pb). Plant material can be pelletized with AI,O,. The Se is separated from the matrix together with Cd, Sb, TI and Bi only, and can be finally eluted with HNO,/HCI = 111, or with 2M acetic acid/20% H,Oz = 7+1 [61,62,63]. After neutron activation and burning of the sample in oxygen, selenium can also be retained in a cool trap, adsorbed upon active carbon in a stream of nitrogen, and finally counted [64]. The oxygen necessarry for burning can also be activated electrically under reduced pressure, which is called low-temperature ashing. Because the sample boat is not heated directly, selenium remains in the ash, which can be dissolved easily in mineral acid. The method has been tested for plants and soils, as well as for coal and coal gasification products [45,65].

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- 2. METHODS FORSEPARATION _ANDSPECIATION O F S E L E N I U M

2.1 GAS CHROMATOGRAPHY

2.1.1 PIAZSELENOLS

Se(IV) slowly reacts with o-phenylenediamine and its substituted derivatives (CI, Br, NO,) to form piazselenols, i.e. 5- membered rings with two carbons, two nitrogens and one selenium, at pH 1-2.5 [ 5 9 ] , which can be extracted into toluene (benzene) and determined by GC. Substitution of o-phenylene-diamine in position 4 influences the extractability of the reaction product with Se(IV) into toluene in the order H<NO,<CH,<CI, and the gas-chromatographic sensitivity on a thermal conductivity detector in the order CH,0<CH,<N02<CI<H [50,66]. The benzo- and nitro- derivatives are the most thermally stable [67]. 4,6- dibromopiazselenol allows a detector temperature of 280" [68,69]. Nitric acid does not interfere with the formation of 5- chloropiazselenol, but oxidizes the reagent. The oxidation products are, however, not extracted into toluene [SO]. Excess reagent can be removed from the toluene layer in a further cleaning step, either with IM- NH,, or with dilute perchloric acid [69], if necessary. Separation is run on non-polar sorbents within a range of 170-200". As the column material, for the 4-nitro- derivative 4%SE-30 + 6%0V-2 10 on 80/100 Chromosorb W-HP, 3% ECNSS-M on 60/80 Gas Chrom Q, and 3.8% SE-30 on 801100 Gas Chrom Q have been compared. In all cases, the retention time lies within the range of 2-5 min [17]. The retention times of fluoro- piazselenols are shorter than for all other derivatives, but, with the ECD, their responses differ widely [67]. For detection of the analyte, electron capture detectors are favourable. The determination of the 5- chloropiazselenol with a thermal conductivity detector is also possible, but less sensitive [50]. In a flame photometric detector, piazselenols, like other Se containing compounds, respond to a broad system of bands likely due to Se?, with dominant eniissions between 450 and 500 nm. Addition of I % methane to the carrier gas extincts interfering peaks from sulfur containing compounds [70] As element- specific detector, a microwave- plasma has been also used [71]. The GC- determination of Se as piazselenol- derivative allows the determination of down to Ingig after decomposition and enrichment steps in biological and metallic matrices [ 1739 72,731. The advantage over common photometric measurement of the coloured compound lies both in its extractability, contrary to the compounds of the reagent with Fe(III), Cr(III), Sn(IV), V(V), CU, Ag, Pb, Hg, AS, Sb, Bi [5O,S9,67], and the separation from excess reagent. For dairy products, however, it is advantageous to remove extractable unknowns (lipids?) by shaking the sample with toluene prior to addition of the reagent [ 191 or to decompose the sample with Mg-nitrate [22]. Determination methods of piazselenols have been already intensively reviewed till 1984 [741.

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2.1.2 VOLATILE COMPOUNDS

By means of gas-chromatographic separation and detection, alkyl selenium compounds, phenyl selenium compounds, selenium fluoride, selenium hydride and selenium trimethylsilyl derivatives can be analyzed Trimethylsilylselenide can be obtained from selenate in diniethylformamide Selenium hydride and substitutes are the result of the reactions of selenite and selenonium compounds with NaBH,, finally detected by AAS (see "hydride- AAS") Alkyl selenium compounds are major metabolites in biological systems GC offers the possibility of the direct determination of the most volatile of them, without derivatization Among them are dimethyl selenide, dimethyl diselenide, diethyl selenide, diethyl diselenide, di-n-propylselenide or diselenide, ethyl seleno cyanate, diphenylseleniurn, Se-acetophenone derivative, or carbon diselenide Element- specific detection with AAS is preferable, because the chromatogranis ale much simpler Quartz- tube atomization (in presence of some hydrogen), as well as graphite tube atomization have been used The deuterium background correction of the AAS at 196 I nm has sometimes difficulties with non-specific molecular absorption by various organic impurities, but addition of ca 10% hydrogen to the argon carrier gas circumvents this problem, and, moreover, doubles sensitivity [75 ] For the determination of volatile Se- species in ambient air, pre- adsorption upon silicone oil on Chromasorb W [ 7 6 ] , or cryogenic trapping with liquid nitrogen [75] can be used The stability of dimethyl diselenide decreases above 140". therefore, flash evaporation with pre-heated carrier gas was applied By GC with Se- specific detection and pre- adsorption, the formation of dimethyl selenide and dimethyldiselenide gases evolved by astralagus from given sodium selenate i n a greenhouse, could be monitored [76]

2.2 LIQUID CHROMATOGRAPHY

Within this chapter, all methods of separation and enrichment of Se- containing species from a liquid phase on a solid packed on a column, are treated Column methods are used both for enrichment and species separation The time needed for separations is rather long, but in many cases quantitative detection on line saves the time for the application of another method for final determination

2.2.1 INORGANIC SORBENTS

Aluminum oxide adsorbs both Se(V1) and Se(1V) from acidic solution I n presence of 1 M- phosphoric acid, only selenite I S adsorbed. together with Sc, Ta and the rare earths I n case of neutron activated samples, Ta interferes with 75-Se counting and has to be precipitated with inactive carrier prior to the load of the column [77]

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2.2.2 USE OF CHELATING ANCHOR GROUPS

Multielement reagents or rather specific groups are fixed on a solid, which is finally packed to a column to pick up the wanted species from a large sample volume, or from an unwanted matrix. In case of selenium, only few attempts of synthesizing home-made column packings are reported. From weakly acid solution, dithizone or dithiocarbamate on active carbon take 60-90% of Se [78]. Bismuthiol-11-sulfonic acid loaded on an anion exchange resin has been used for the enrichment of Se prior to HPLC [79], but it can be operated also in a simple column technique, with penicillamine/cysteine as eluent [SO]. On DEAE- cellulose, a weakly basic diethylaminoethylcellulose, selenate IS strongly adsorbed from dilute formic acid (0.1-IM), whereas selenite passes the column. Selenate can be eluted with 0.05-1M HNO, [81]. CI- decreases the adsorbability of selenate and should be less than 0.01M, which is important for the enrichment from sea water [81].

2.2.3 REVERSED PHASE HPLC

The detection limits in HPLC recently approach AAS and electrochemical techniques.

2.2.3.1 Derivatization To achieve either optimum sensitivity, or multielement capabilities, products with known reagents are chromatographed. As they commonly start from Se(IV), information about speciation depends on a prior separation. Various piazselenols, which result from reactions with selenite, and which are widely used for spectrophotometric, fluorimetric or gas-chromatographic determinations, can be se- parated from excess reagent on Bondapak C18 or Unisil 5C18 [82] by HPLC. Thus, the reaction product of diaminonaphtalene with selenite has been extracted with cyclohexane, chromatographed with acetonitrile as eluent, and finally detected fluorimetrically [82]. The naphtylpiazselenol can also be chromatographed in chloroform on Nucleosil 10, containing amino groups [83], or in pure methanol on C18. Fluorescence detection is more selective at 480nmheading at 580nm, because the reagent is not excited. A detection limit of 0.15ng in 100 p1 extract has been achieved [84]. The 5-nitropiazselenol and the 5-chloropiazselenol can be easily separated from excess reagent on Nucleosil I 0-C 18, with methanol/water as mobile phase. The Se- carbamates cannot be used for photometric measurement, because of co-extraction of other coloured compounds, and adsorption of the excess reagent. For the carbamates, an UVNIS detector, operated at 254nm, is sufficient [83]. On LiChrosorb RP-8 and Nucleosil 10-C 18 it is possible to separate various diethyldithiocarbamates of Se and other heavy metals, obtained from extraction at pH4 with chloroform. For Se, methanol/water = 7+3 is preferable over acetonitril/water= 6+4, because of overlap of Se with Pb in the latter eluent [85]. Chelates of Se with APDC (ammonium pyrrolidin dithiocarbamate) have been enriched from HWpH 1.2 at C18 bonded silica gel, subsequently eluted with methanol and finally determined by graphite furnace AAS [86]. Another promising derivative has been gained from the reaction of Se (IV) with penicillamine, which is selective in acid solution. The product can be chromatographed on

41 1

Capcell Pak C18 in acetonitnleiwaterlphosphoric acid = 400 600 1 and detected by UV, but much more sensitive by derivatization to a fluorophore by reaction with 7-fluoro-4-nitrobenz-2, I ,3-oxadiazole [87]

2.2.3.2 No derivatization Selenate is adsorbed on CIS reverse phase Altex Lichrosorb RP-I from 0.001M hexadecyltrimethylammoniumbromide pH9 5 and thus separated from selenite, which passes the column. Selenate can be eluted with methanol. Detection with Zeeman- AAS allows the separation of various low-molecular As and Se species simultaneously [88]. Similarly, As and Se species have been separated without derivatization in acetate buffer on nucleosil, and detected by sequential ICP. Selenate is strongly retained, and eluted with ammonium phophate buffer pH 6.9 [89]. Selenols RSeH, diselenides RSeSeR, and selenylsulfides RSeSR have been separated on a 3pm diameter particle size Biophase ODS column, in 0.05M NaH,PO,/ 5% acetonitrile/0.004% Na-octylsulfate. An electrochemical detector with dual HgiAu amalgam electrodes allows to distinguish between the 3 groups because of their different electrochemical reactivity. This enabled the determination of reduced and oxidized glutathione in blood plasma [92].

2.2.4 ION EXCHANGE AND ION CHROMATOGRAPHY

As selenium is anionic, it passes the Dowex 1x8 cation exchanger in dilute HCI, whereas e.g. Ag, Au, Cd, Hg,Zn are adsorbed [91] The cation- exchange resin 1RA-200, charged with 3M- HCIO,, has been used to separate Se from various cations prior to determination by anodic stripping voltammetry, because Se is not retained from > 0.05M- HC10, [92]. Selenite and selenate can be separated at many anionic exchangers. For selenite, the sorption generally increases from sulfate form to chloride form to hydroxide form, and for selenate it is just reverse. Versus pH, selenite has a steep sorption maximum at pH 3-4, weak sorption at pH>6, but none beyond pH2. Contrary to this, selenate is strongly adsorbed at pH<2 [93]. In dual column configuration, with separator and suppressor column, in carbonate buffer, selenate is slower than the common anions, even sulfate, but without interferences. It is also possible to separate selenite, which comes between phosphate and nitrate, by raising the pH with additional carbonate, but the time for each chronlatogram of 36 min seems too long for practical work. As the alternative, oxidation of selenite and elementary selenium with H,O, to selenate is proposed [94]. Among the 0x0-anions, however, selenate is fastest. In 0.003M- sodium carbonate, selenate elutes in only about 6 minutes, followed by tungstate, molybdate, arsenate and chromate, which can be all gained by treating the sample solution with hydrogen peroxide. In this case, an excess of As over Se of 1 : 1000 is tolerable, detection limit of 1 pg/l without column-preconcentration have been achieved WI. In 0.002M- Na$03/0.002M- KOH, selenite is fairly resolved from neighbouring CI and nitrate, and selenate from neighbouring sulfate and phosphate [96]. With a single column device, in phtalic acid/formic acid pH 2.7 on Vydac 302, selenite as the slowest was separated from chloride, nitrite, nitrate and phosphate in soil extracts. At pH 4.5, however, chloride, phosphate and nitrite interfered with selenite. Sulfate and selenate did not elute from the column [97] at this pH When the column was eluted with KH-phtalate buffer pH6.5, the broadening of selenate could be depressed by use of the

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sulfate form. Upon the conventional conductivity detector, the sensitivity of selenite was only 1/10 compared with nitrate [97,98]. For this reason, the eluate was converted to the hydride on-line and fed into an ICP via a gas- liquid separator. The conversion to the hydride was reported to be 100 times more sensitive than direct nebulization of the eluate WI. For the detection of selenite in single column configuration, under optimum conditions, maximum excess of other anions up to 10000 is possible. It is, however, advisable to remove high chloride levels (analysis of sea water) by reaction with a Ag- saturated cation exchange resin [97]. For selenate, in phtalate buffer 4.6, the resolution selenate to sulfate is possible up to 80 mg/l sulfate, but the calibration of selenate is sulfate- dependent [98].

2.3 LIQUID-LIQUID EXTRACTION

Nearly all procedures start from Se(IV), separating it from other matrix elements, or in a few cases from selenate. For the extraction of selenate into a non-aqueous phase, only dioctyltin-dinitrate in CHCIJTBP = 1+3 could be found, No method has been reported so far for the separation of organoselenium compounds, but from this review an idea about the possibilities for the formation of lipophile Se-containing compounds might be achieved.

2.3.1 EXTRACTION FROM HYDROCHLORIC ACID

The extractability of Se into benzene, chlorobenzene, dichlorobenzene or nitrobenzene from HCI is generally poor, but high distribution ratios are observed with binary mixtures of 9M- sulfuric acid and acid halides [loo] Separation of many other elements is possible, S b is coextracted [ 1001 From 6M- HCI, Se is not extracted into diethylether or diisopropylether, and can thus be separated from Au,Fe, Ga, Mo, Sb(V) and TI(II1) [ l o l l Similarly, it does not move into MIBK from 7M- HC1/7M-LiCI, contrary to As, Au, Fe, Ga, Mo and TI(II1) [ l o l l From concentrated hydrochloric acid, Se can be quantitatively extracted into toluene [ 1021 In concentrated HCI, selenate is converted to selenite and cannot be detected per se

2.3.2 EXTRACTIONS FROM HYDROBROMIC ACID

Toluene quantitatively extracts Se from > 4M- HBr, and chlorobenzene at >6M- HBr, but benzene takes only 60% at maximum, from concentrated HBr [ 100,102]. Quantitative separation from many elements can be, however, achieved from 9M- H,SO,/O.lM- HBr into dichlorobenzene [ 1001. Diethylether takes only 31% of Se from 6M- HBr, but none from dilute solutions (c3M). Thus, in IM-HBr, separation from Au and TI(III), and in 0. IM-HBr, separation from Hg can be achieved [101,103].

2.3.3 EXTRACTIONS FROM HYDROCHLORIC ACID WITH THE AID OF UNSA- TURATED HYDROCARBONS

Unsaturated hydrocarbons react with selenite to compounds of the type (RCHCICH2)2SeCI,, which are extractable into chloroform or methylene chloride [ 1041.

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2.3.4 REAGENTS WITH N- CONTAINING GROUPS

0- phenylendiamine and its derivatives are widely used for the isolation of Se The extract can be subjected to gas chromatography or liquid chromatography (see chapters l,2), but also simply for extraction- photometry, or extraction and gamma-counting after neutron activation The reaction IS very slow, and requires I hour at room temperature, or 10 miri at 75" [ 1051 N- phenylbenzohydroxamic acid can extract Se from 7M- HCIO, into chloroform as a yellow complex, utilizable for spectrophotometry (345 nm) or AAS [ 1061 With tri-isooctylamine, however, Se is not extracted into 4M- HCI, and can thus be separated from Au,Ga,Os,Re, and Cd [ I0 I ]

2.3.5 REAGENTS WITH 0-CONTAINING GROUPS

Selenite reacts with excess aldehydes or ketones to compounds, extractable into chloroform from > 4M- HCI solution [108,109] With 30% TCMA (trichloro-methylacetate), Se can be selectively extracted from 4 5M- H,SO4I7M- HCI into xylene, it is accompanied only by Pu, Np(IV), Sb(1II) and Co, and used for radiochemical separation The extraction got enhanced with the addition of water- miscible organic solvents to the extraction system [ I091 2 3 6 REAGENTS WITH P- CONTAINING GROUPS

The affinity to P-containing groups is low Into undiluted tributylphosphate. Se I S not extracted up to 6M- HCI With TBP, in IM-HCI, separations are obtainable from Au,Ta, and TI(III), and in 6M-HCI from Au,Fe, Ga, Nb,Pa,Sb,Ta,Te,In,Mo and TI[ 1011 With TOPO, separation of Se from 6M-HC117M- LiCl has been described [110]

2.3 7 REAGENTS WITH S- CONTAINING GROUPS

With diethyldithiocarbamate ( - DDTC), extractable compounds are formed in acid solution, but this competes with rapid decomposition of the reagent From 2M- HCI or 2M- H,SO,, probably a mixture of Se(DDTC), and Se(DDTC) is extracted [ I I I ] , together with Cu,Ag,Hg,TI(III), As(III), Sb(III), Bi,Te(IV), Pd and Pt Se is not extracted from pH7 into CCI, and thus separated from Cu,Ag,Zn.Cd,Hg,In,TI,Pb,Sb,Bi,Te,Mn,Fe,Co,Ni,Pd and Pt [ 1 1 I , I 121 From acetic acid pH 2 6, Se and A s are extracted with DDTC [ I 131 Se I S

also extracted from acetate buffer pH4 with diethvldithiocarbamate into CCI,, the selectivity can be improved by masking acconipanving cations with EDTA To prevent decomposition of the reagent, DDTC is added to the neutral sample solution, and the buffer is added at last [ I 141 Corresponding to the final AAS- sgnal, MIBK takes much less Se than CCI, Selenate does not react [ 1 141 In 0 1 M- sulfuric acid and citrate buffer pH5, the reactivity with DDTC of Se is poor [ I IS] When ammonium pyrrolidin dithiocarbamate (APDC) is used instead of DDTC, the subsequent graphite-furnace-AAS signal is lowei. maybe because of volatilization from the tube. but APDC is less pH- dependent for Se [ 1 14, I I61 0- isopropyl-N-ethylthiocarbamate possesses very high selectivity for Ag and Hg from HNO,, H,SO,, HCIO, and HCI, whereas Se is not extracted into chloroform from samples containing no bromide [ 1 171

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The reaction with dithizone is incomplete and cannot be used for the isolation of selenite; however, if a pH more than 1 is used, other cations can be removed from the selenium containing sample [ 11 4,1181. In graphite-furnace-AAS, the Se-dithizonate is volatile and yields nearly no signal [ 1141. With K- ethyl xanthate, Se(IV) is quantitatively extracted from 0.1-IOM-HC1, and Se(V1) above 7M; selenate may be converted to selenite in HCI. The separation is rather selective. From 2M- HCI into chloroform, Se is only accompanied by Cu,Ag,Au,Pd, As,Bi,Mo, and partially Pt,Sn,TI and Fe. From 1M- HCI, only Ag, Pd,As, Mo, beneath Se move quantitatively into the extract [ 119,120]. During the extraction of Mo-ethyl-xanthate into a mixture of CC14henzene or into chloroform from 1 .SM-HCI, Se is coextracted [121]. In these complexes, Se is presumably bivalent [122]. The co-extracted Mo does not interfere with the spectrophotometric determination with 3,3' diaminobenzidine [ 12 11. Xanthates allow separation of selenium from matrix Cu,Ni,Pb and Zn [121]. With K-butylxanthate, Se(1V) yields a 1.4 complex, which can be extracted into CC14 at pH 2.0-4.7 and used for spectrophotometric detection at 395nm [122]. If benzazoles contain a mercapto group, like 2-mercaptobenzthiazole or 2- mercaptobenzoxazole, Se(IV) quantitatively passes into the organic phase from 4- 1 OM- HCI (chloroform). The use of chloroform permits the separation Se-Te, the equilibrium is reached in 5-45 min, depending on the HCI- concentration. The solvent does not participate, because curves obtained with different solvents are analogous [ 1231. By means of 0.2M-di-n-butyldithiophosphoric acid/CCI,, Se is extracted in the range 0.03-9M- HCI and 0.01 5-2.5M- H,SO,. A total separation from Fe, Ga,Os and Te over the entire range is achieved. In 0.1M- HCI, there is no separation from Cd, In, Mo, Ni, Sn(II), TI(II1) and Zn; in 5M- HCI, for example, there is no separation from Ag,As,Au,Bi,Hg,In,Pb, Mo, Pd, and Sb [101].

2.3.8 SEPARATIONS WITH ORGANOTINS

In the range pH 2-6, compounds of e.g. triphenyltin, trioctyltin and others, with Se(1V) and Se(V1) move into chloroform, MIBK, octyl alcohol; if the non-polar solvents benzene, xylene or cyclohexane are used, TBP (tributylphosphate) or TOP0 (tri-octylphosphinoxide) are added. Optimum conditions have been achieved with 25% TBP in chloroform or o-xylene [124].

2.4 COPRECIPITATION AND SORPTION METHODS

A solid collector is either added to the sample solution, or produced in the sample solution by precipitation of a carrier, and finally separated by filtration or centrifugation. This leads to a uniform solid matrix, which can be either dissolved further, or directly submitted to XRF or gamma-counting after activation. Concerning speciation, in many cases all forms of Se might be precipitated, which is an advantage, if total contents are wanted. Hydroxides, however, rarely adsorb Se(V1) [125,126] (compare: movement of Se in soils).

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2.4.1 PRECIPITATION AS ELEMENT

Reductive coprecipitation with elemental tellurium or elemental arsenic is very selective, getting both selenite and selenate [127]. Huge excess of oxidants in the sample, e.g. nitrate or chlorate are better avoided, because they consume much reductant. To ensure the presence of excess reductant, iodide can be added as indicator; but in this case, Cu is coprecipitated as iodide and should be removed, when it is present in the sample above the normal geochemical level [ 1281. Se is coprecipitated with Aso from arsenite and hypophosphite in 6M- HCI during 20-30 min boiling [129,130,131]. Similarly, tellurium can be precipitated with sulfite from HCI/HBr [ 1321, with hydrazinium sulfate from 3M-HCI [42] or 4M-HCI [ 1271 or NaOH after the centrifugation of other hydroxides [ 1331. Active carbon has been used as a sorbent for selenium, either after reduction of selenite with ascorbic acid, or, to get total selenium, after reflux with thiourea [134,135]. Also, hy- drogen selenide, obtained by reduction of the sample with Zn" or NaBH,, adsorbs on active carbon, whereas selenite and selenate do not [ 1361 Elemental Se is rapidly adsorbed upon pyrex glass and polyethylene [ 1341; precipation, centrifugation, decantation and dissolution without change of the vessels is therefore preferable.

2.4.2 COPRECIPITATION WITH HYDROXIDES

Coprecipitation with hydroxides separates Se together wit other trace elements from alkali, alkaline earths, or large excess of unwanted anions (e.g. nitrate, sulfate). The hydroxides of Al,In,Ga,Zn and Mn are not suited for the collection of Se [ 1261. Fe- hydroxide sorbs selenite in weakly acid solution (pH 5 [137]; pH 2.4 in presence of ammonium chloride [138]), but not in alkaline solution at pH 8 (bromothymol blue indicator) [135]; selenate is not coprecipitated with Fe- hydroxide [137]. As an alternative to filtration or centrifugation, Fe-hydroxide can be flotated with bubbling nitrogen at pH 3 . 5 - 5 . 3 in presen- ce of Na- dodecylsulfate or Na- laurylsulfate [ 139,140]. Se is accompanied by Ge,As, and Sb. Like Fe-hydroxide, Zr- hydroxide at pH 8 is reported to coprecipitate selectively Se(IV) [141], but not Se(V1). Coprecipitation with La- hydroxide at pH 9-10 separates As,Se,Te,Sb,Bi, Pb,Fe and Sn from matrix Cu prior to hydride AAS [142,143].

2.4.3 COPRECIPITATION WITH SULFIDES

Coprecipitationkorption with sulfides is only useful, when the precipitates need not be dissolved again, e g for final determination by XRF or NAA Se can be sorbed on thin layers of ZnS, MnS, CuS, or PbS at pH 3-6, but high salt loads interfere, like sulfite, thiosulfate, phosphate, citrate, tartrate [ 1441 Precipitation from homogenous solution with thioacetamide has been successfully applied to samples from soil decomposition, after hydroxide precipitation [ 1451

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2.4.4 COPRECIPITATION WITH ORGANIC REAGENTS

In many cases, multielement preconcentration with common reagents IS currently used with subsequent determination by XRF, which requires a uniform solid and low-atomic weight matrix. Precipitation with dibenzylammonium-dibenzyldithiocarbamate at pH 5/pH 4 allows the separation of 12 elements from alkali, alkaline earths and lanthanides from natural water [146,147], prior to XRF. Selenite and selenate are not coprecipitated or sorbed quantitatively on active carbon together with dithiocarbamates, oxinates and dithizonates, [ 148,1491. After reduction with sulfite or thioglykolic acid, carbamates precipitate Se also [ l SO]. Boiling of a 3M-HCI sample solution with a poly-thioether yields a precipitate containing Se and Te; this separation is applicable to ore analysis [ 15 11. Precipitation of polyvinylpyrrolidon + thionalide at pH 4 has been also utilized for the separation of Se and other trace elements from alkali and alkaline earths from natural waters, prior to XRF [ 1521. Cellulose filters wlth immobilized 2,2’-diaminodiethylamine quantitatively sorb selenite and selenate from natural waters in the range pH 3-7, together with other 0x0-anions, like chromate, arsenate, vanadate, molybdate, and tungstate [ 1531. The collection efficiency is strongly depressed with salt (e.g. NaCI) concentrations above 0.01M.

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- 3. METHODS FORDETERMINATION O F S E L E N I U M

3.1 SPECTROPHOTOMETRIC A N D FLUORIMETRIC DETERMINATION METHODS

In many cases the same reagents can be used either for spectrophotoinetric or fluorimetric determination of selenium In comparison with other determination methods, the detection limit is not much inferior at all, especially if larger sample weights are used Strong oxidants and reductants react with the reagents, and thus interfere To increase selectivity, solvent extraction as separation step IS often included If not stated otherwise, after wet decomposition with oxidizing acid mixtures, the sample has to be brought to Se(IV) with HCI

3.1 1 AROMATIC 0-DIAMINES

Aromatic 0- diamines react with Se (IV) in acid solution to yield coloured and extractable compounds with a 5-membered ring containing one Se, two N and two C- atoms They are called piazselenols [ 1541

3.1.1.1 0- Phenylenediamine and derivatives The unsubsituted o-phenylenediainine [43,155] as well as derivatives substituted at position 4 and/or 5 with methyl-, chloro- [55,83,156] and nitro- groups [83] have been added to the sample in 0.1-0.5% aqueous or 0. I M-HCI solution For the reaction velocity, there is a pH optimum, usually pH 1-3 [ 15.51. An overview of acidity constants of reagents and products has been given in [ 1541. Reaction conditions of pH I -3/50° [ 1551, pH 2/30 min/2O0(low salt contents)[43], 2h/20°[S5] and 10 miiii40" [ 1561 have been reported. The reaction product can be extracted into chloroform in the range pH 1 7-12.5, and moves only partially back into the aqueous phase with NaOH [ISS]. To avoid coextraction of the unconsuined reagent, however, extraction at pH 2 is preferable. Interfering cations can be masked with EDTA prior to the addition of the reagent [43], or Se is isolated from the matrix by volatilization of SeOZ [43] or SeBr, [55]. Photometric determination has been exerted of the toluene extract (335nm for the unsubstituted [43], or 34 I nm for the 4-chloro- derivative [SS]. A clean-up of the chloroform- extract by HPLC prior to photometric determination at 340nm [83] or fluorimetric determination at 550nm [ 1561 on-line, very much improves the detection limit, because of the separation of the proper signal from the reagent and by-products [83] For HPLC, Nucleosil C 18/chloroform [83], and LiChrosorb RP-8imethanol-water 80-20 [ 1561, have been used. The piazselenol of the N-phenyl-substitute of o-phenylene-diamine, called 2- aminodiphenylamine, is obtained by reaction 2h/2S0 or 1 h/40", and is extracted as the ion-association-complex with perchlorate (from 3M- perchlorate) into hexanol-chlorobenzene = 6.4. With respect to other phenylenediamines, the higher molar absorption coefficient (18000 l/mol.cm) allows to reach a detection limit of 0 5pg Se/lOml sample solution [ 1571.

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3.1.1.2 2,3- Diaminonaphtalene This reagent has been introduced in the sixties [ 158,1591 and successfully used since then. Usually a 0.1% solution of the reagent in 0.1M- HCl is prepared, but also a 0.1% solution in 5N- H,SO, has been reported [ I 11. Stored in the dark under cyclo-hexane, the reagent is stable for two weeks [28], but most authors prepare the reagent solution daily. Before the reagent solution is added, excess oxidants (e.g. NO,) can be expelled by heating with formic acid [28,37,121], or addition of hydroxylamine [ 1 1,25,28,37]. Interfering ions, like Fe and Al, have to be masked with EDTA, EDTA/NaF [160], or EDTA/NH,F [34]. The adequate masking capacity of EDTA towards Fe is in the mole ratio 1 : 1 [28]. Separation from matrix Fe can be achieved by coprecipitation with Te [160], by ion exchange [80,126]. Matrix Sb can be masked with tartaric acid [34]. Matrix Sn and Cu still interfere [ 1621. As the reaction and subsequent extraction are rather strongly pH-dependent, the pH has to be adjusted exactly. This is done with ammoniahydrochloric acid [25,28,34,121], or sodium acetatehydrochloric acid [ 1601, and can be facilitated with glycine [27]. Optimum pH range is 1.8 - 2.0, but at pH 0.4 still 85% of the Se are extracted [161]. More troublesome seems to be the co-extraction of the reagent and others at higher pH; when pH 2.4 was chosen, the fluorescent peak had to be corrected for its background [27] The resulting piazselenol is extracted uniformly with cyclohexane or n-hexane [34]. After excitation at 365nm or 378nm, the fluorescence of the piazselenol is measured at about 520nm. As utmost detection limit, 2ng/g (for 1 g sample/ enrichment into 5ml cyclohexanel no aliquots) [34] has been reached. The detection limit can be significantly lowered to 0. I 5ng/20pI by HPLC- separation of the piazselenol on C 18 reverse-phase column in 100% methanol [84], or on Bondapak C I8 and Unisil 5C18 in acetonitrile [ 1631, with fluorimetric on-line detection. This removes several chemical species which rise the blank level of the fluorimetric determination The procedure, including reaction, extraction and measurement, could be successfully transferred to an automatically working segmented-flow system [ 1641

3.1 . I .3 3,3'-Diaminobenzidine Reaction and interferences are comparable to diaminonaphtalene and the o-phenylenediamines. The reagent is used in 0 2-0.5% freshly prepared aqueous solution, and reacted with the sample at pH2-3 [ 126, 155,165,1661, for 50min at 45' [ 1551; alternati- vely at pH 1.25 for 45 min at ambient temperature (1211. The pH is adjusted with ammonia, oxidants interfere. For the extraction of the resultant selenium compound into toluene or chloroform, pH has to be increased to pH 5-10, optimum is pH 6.5 [121,155]. Photometric measurement is done at 420nm, and fluorimetric measurement at 570nm. Large excess of Fe, Pb, Cu were removed by extraction as the xanthates into chloroform, or by ion-exchange, minor amounts can be masked with tartaric acid and EDTA [ 12 1 .I 261.

3.1.2 S- CONTAINING LIGANDS

Many S-containing ligands react with Se, like diethyldithio-carbamates, dithiophosphates, toluene-3,4-dithiole, dithizone [ 1551 The selectivity, however, is poor, which often prevents their use for the analysis of real matrices 2-Mercaptobenzimidazole selectively and rapidly reacts with Se in 2M- H2S0,, and can be extracted as ion-associate into chloroform/isopentanol = 8 2, which is applicable to the

419

direct analysis of steel- decomposition solutions, -= 10400 I/mole cm at 33Snm Fe, Cr, Co,Ni,Co,Mo are not extractable without HCI, only Cu, Te and Bi interfere [162,167] 4,5-Diamino-2,6-dimercaptopyrimidine yields a pink colour with Se(1V) in 0 5-2M HCI, and does not need to be extracted into an organic phase As detection limit, 0 01 pg/ml could be achieved (3-fold of diaminobenzidine), and applicated for semiconductors and animal feed premix, Au, V, Fe, and Cu interfere [ I681 Methiomeprazine hydrochloride forms a blue-coloured species with Se(IV) in 9 9 - I 1 2M- H,PO, in about 40 min, with a very high -=29830 Vmolecm at 644nm, from 23 ions investigated, only Au interferes [ 1621

3.2 DETERMINATION OF SELENIUM BY ATOMIC ABSORPTION METHODS

3.2.1 FLAME -AAS

The low levels of occurrence usually encountered, as well as the poor performance of the technique limit the application of flame-AAS to quality control of Se- containing products as well as sulphidic ores The main atomic line for the determination of selenruni is 196 03 nm At this short wavelength, absorption from molecules, and light scattering from oxygen as well as from carbon- containing gases and water vapour cause severe noise of the zero-line [ 169,1701 In the NIO/C,H, - flame, the detection limit is about 2 pgiml In the argon- or nitrogen- entrained hydrogen-flame, the sensitivity is 2-2 5 fold higher than for the conventional acetylene /air flame [75,169,171,172], low gas-flow is favourable The Ar (or N,) is entered like air, and H2 like fuel to the burner system, and the auxiliary oxidant entrace IS closed Similar constructions have also been used successfully as element- specific detectors in gas- chromatography to detect gaseous Se- compounds after separation on column in a stream of inert gas [7S] The exit of the GC can also be arranged concentrically around a hydrogen flame, which burns within a quartz cuvette, mounted in the optical AAS- path (see further hydride methods/flame-in-tube techniques, [76]) Another possibility to achieve reasonably low background absorption, is to use the "Slotted tube atom trap" A quartz tube is mounted on the burner- head in that way, that the optical pathways lies within the tube axis, and the flame is led through two slots in this tube This lowers the detection limit for the determination of Se in flame by a factor 3-6, irrespective of the equipment and the geometry used [ 170,173,174] Use of an ultrasonic nebulizer and a heated mixing chamber instead of conventional pneumatic nebulization results in only minor improvement of the detection limit in the H,/air - flame [ 1751 Fe and phosphate (as P,-bands) can cause spectral interferences in real samples, resulting in a negative D2- compensation signal Separation from matrlx Fe, P and salt matrix, or use of Zeeman - background- compensation is thus recommended [ 1761 Direct aspiration of combustible organic solvents (esters, ketones) causes much more noise in their flames than from an aqueous phase, and only 20-30% of the analytical signal IS

obtained [ 169,1771 Se could be selectively extracted from sample solutions from ores, Cu and Pb concentrates in HCI with 5% acetone into methylmethacrylate, and diiectly measured in the organic phase Only Au and Fe go along with the Se, and determinations in the pgig range are possible [ I 771

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If the sample can be atomized directly from a Pt- loop rapidly inserted into the analytical flame, detection limits can be greatly improved by electrolytical deposition of the analyte upon this Pt- loop beforehand. Above all, this separates from a salt matrix, and especially from Fe and P, which are likely to yield non-specific signals. It is important to get the dried Pt- loop into a position of reproducible geometry, to cope with signals of the distortion of the flame itself. Thus, Se has been determined in sea water, brines, technical sulphuric acid, and biologcal samples [34,171,172,178,l79). If an AdH, - flame is used with the mentioned technique, a 10-fold improvement of the detection limit with respect to the C2H2- flame is achievable, but electrical pulse- heating of the Pt- loop is essential Because the electrolyte contents and coprecipitated Cu influence the electrolysis step andthus the analytical signal, standard addition is recommended. For biological samples, 0.05 pg/g has been achieved as the detection limit [31,171,172,179].

3.2.2 HYDRIDE METHODS

In hydride methods, selenite in an acid sample solution is reduced to H,Se and separated from most of the matrix via the gas phase (The hydride forming reaction may also cause the transformation of Se-containing organics to compounds of enhanced volatility, whlch may be of use for speciation studies of non- digested samples, in combination with gas- chromatographic separation) The resulting H,Se can be atomized either in a11 H,- entrained flame, in a heated quartz tube, or a heated graphite furnace The Se- atoms are usually measured in the absorption mode, but plasma emission and atomic fluorescence methods have also been applicated

3.2.2.1 Hydride generation In acid solution, NaBH, decomposes within 10 msec, but the reaction to form hydrides is usually faster [180]. Generally, As, Sb, Bi, Se, Te, Ge, Sn and Pb yield volatile hydrides upon reduction, which are subsequently atomized by thermal decomposition. As only quadrivalent Se reacts to H2Se, Se- containing organic compounds have to be decomposed, and hexavalent Se has to be converted to yield all Se in the quadrivalent form Dimethylselenide and dimethyldiselenide are sufficiently volatile to be stripped from an aqueous sample with only He as the stripping gas, bot 0,-free conditions are necessary [181]. For determination of total Se, the H,Se formed is completely recovered from the reaction solution at pH < 0.7. The decline of the signal with increasing pH was less than calculated from the dissociation constant of H,Se. At pH > 6, no signal is obtained [ 1421. Interferences in the hydride generation step are either due to consumption of the NaBH, -reagent due to oxidants or catalytical decomposition, the reaction of the H,Se formed with other species in the reaction mixture, or just foaming, which prevents the hydrides to be swept out into the atomizer, The amount of interferences largely depends on the geometry of the device as well as the reagent and acid concentrations used Concomitant organics present after insufficient mineralization may cause intense foaming, which interferes in both modes of hydride generation; it flattens the peak and decreases the signal height. As H,Se is readily soluble in water, wet connections to the atomizer severely lower the signal.

42 I

3 2 2 1 I Hydride generation in batch In batch methods, the acid sample solution is put into a reaction vessel, NaBH, reagent solution is added, and the hydrides together with the hydrogen formed are swept into an atomization device As the sample volume added into the reaction vessel can be varied widely (20 pl - 20ml), a dynamic range of 4 orders of magnitude is achieved Peak area measurement compensates for different speeds of hydride generation, caused by different acidities of the samples, oxidants, or foaming A1 powder [ 1831, SnCI,, and Zn powder [ I841 also yield H-Se, but stripping from the reaction solution is insufficient In 0 3M HCI, TiCI, reduces Se(V1) to Se(1V). but it does iiot react with Se (IV) further

Nowadays, an about 3% NaBH, / I % NaOH aqueous solution is used for batch- hydride generation in most cases Stripping of H,Se from the reaction vessel into the gas phase sufficiently rapid Hydride generation is possible from 0 3 - 6M- HCI [6] Generally, the amount of tolerable interferents is higher at higher HCI- concentrations because of the formation of chloride complexes on the one hand, and more rapid generation and stripping off on the other Within the range I M - 6M HCI, the same peak area had been achieved, but peak height in 6M- HCI dropped about 30% [ 1861 due to dilution with the H2 generated Sweeping the hydrides in a previously evacuated atomization device can improve the performance of Se in the peak-height evaluation, more than in case of Sb, Sn and As [ 1871 Hydride generation from H$O, IS possible within the range I-10M [ 1881, but Se blanks in H?SO, have to be considered [6] Hydride formation with 3% NaBH, in batch tolerates up to 2M HNO, [ I891 But if only I% NaBH, is used, HNO, extincts the signal even as low as I 5%, Co, Cu, Ni, Ag and Sn

Fe IS reduced to Fe(I1) and thus consumes reagent I t can be masked with SCN-, citrate, EDTA, I ,lo-phenanthroline and others Reduction of Fe(1ll) prevents the formation of elemental Ni, which catalyses the decomposition of H,Se [ I891 Co, Ni, and Cu catalyze the decomposition of NaBH, in acid solution They have to be masked with halides or other stronger coniplexing agents KI increases hydride formation, decreases the NaBH, decomposition, and precipitates Cu as the iodide This leads to a significant increase of tolerable Cu level in presence of k1 [I801 As a proof for the catalytic action of NaBH, decomposition, and against H,Se rection, H,Se could not be absorbed in AgNO, or in Ni(NO,), - solution [ 1841 Kl is preferably added to the NaBH,- reagent, because it slowly reduces Se in acid solution [ 190,191] KI also masks excess Hg, which lowers the signal [I911 Cu can be masked with thiourea [47], and N i with a nearly saturated solution of citric acid [I921 Cu, Ni, and Co interferences can be overcome by addition of EDTA [I931 Other hydride forming elements consume NaBH, reagent, but they rather interfere in the atomization step rather than in the H2Se- formation itself H,Te is metastable and hardly got in the batch technique PbH, is got from the quadrivalent state, which IS usually not encountered in the samples, especially after conversion of Se(V1) to Se(1V) In batch method, addition of Te even improves the Se determination, because because CuTe I S

precipitated, rather than H,Te metastable is evolved [ I941 Precipitation of elemental Te does not interfere with the Se- determination, but elemental Bi strongly does [ 1951

[1851

[1901

422

Bi, Ge, Sn and Pb matrices can be masked by addition of additon of EDTA. Citric and tartaric acid do not improve Se in presence of excess Sb and Tef1941. Pb and Hg at ambient levels are of no influence. Nitrite interferes in the determination via the hydride, because H,Se and NOz- react in acid solution. H,Se is formed, and further reacts with nitrite in the reaction solution before it can leave the hydride generation device. H,Se is absorbed in a nitrite solution at about 88%. At nitrite concentrations below 0.2 pM, standard addition is sufficient, otherwise nitrite can be masked by addition of a 2% solution of sulfanilamide [ 1961. Similarly, addition of an aqueous solution of NH,OH.HCI prior to the hydride forming reaction prevents interferences of nitrite and dissolved nitric oxides [ 1971, which are present after any decomposition with nitric acid.

3.2.2.1.2 Continuous hydride generation In the continuous flow technique, low contact time of the generated hydrides with the reaction solution lower the interferences which are caused by secondary reaction of H,Se. In practical work, the standards have to be closely matched with respect of their acid- and Fe(II1)- contents to the samples analyzed. In the continuous mode, on-line separations are possible, like GC separations of organic substituted hydrides, or LC separation of the matrix prior to hydride generation. Even on-line reduction if Se(V1) by HCI- addition and heating has been exerted [ 1981. In continuous flow, Se evolution is not interfered up to 10% HNO, or H2S0, [199]. Hydride evolution from perchloric or nitric acid alone yields only low signals [38]. Hydride selenide generation from acetate/acetic acid buffer led to very unstable signals in continuous flow generation [ 1821. The Nal3H,- reagent has also been added via an anion exchanger in the tetrahydroborate form, generating the hydrides in a heterogenous reaction on line, and transferring the HzSe along with H, through a gas-permeable PTFE- membrane towards a flame -in-tube atomizer [200].

3.2.2.2 Atomization

3.2.2.2.1 Flame-in-tube technique The product of hydride generation is directly introduced into a H,/air flame or a H2/Oz - flame by means of a N,- stream. The extremely soft diffusion flame has to be protected from room air currents by a burner shield equipped with quartz end windows and Pyrex side plates, which improves the precision two-fold. In the flame, H- radicals are formed in a cloud which atomize the H,Se. The shape of the cloud of H- radicals depends on the gas velocities. Interferents accelerate the consumption of H- radicals [24,186]. In the flame, not only H,Se, but also various Se-containing compounds, like dimethylselenide, dimethyldiselenide and others can be atomized in a similar way, which is important in speciation studies, Some Se- containing organicals can be reacted with NaBH, to yield volatile hydrides, which can be detected after separated by GC in a stream of He in an H,/air- flame [181]. After hydride generation in the batch- mode, it is possible to lead the reaction products directly into the N2/Hz/air flame [41,201], or into an Ar-H,/air flame via the nebulizer of an AAS device [202].

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To improve the detection limit, the hydrides collected in a cool trap can be pulsed into an N,-HZ- flame with a stream of N,- carrier gas The composition of flame gases has to be optimized to get into the range, where the peak height is independent of the gas composition [ 1881

3 2 2 2 2 Heated quartz cell Hydrides together with H? are swept into a T-shaped quartz tube, positioned in the optical path of the AAS- spectrometer, which is either mounted on the burner head and heated with gas, or within an electric furnace The inner surface of the quartz plays a decisive part in the destruction of the hydrides, because i t contains a catalytic film of H- radicals [203] Small amounts of oxygen enhance the signal. due to radical formation, but not due to reaction of 0 2 with the SeH, itself [204] 700-800" is regarded to be the optimum temperature for the decomposition reaction to yield Se atoms The Se, - molecule absorbs at 334 nm and can be measured with a D,-lamp Dimerisation takes place at high Se- levels and lower temperatures [205,206] In analytical practice, optimum temperature is

850-950", to get reasonable temperature of the inlet part of the tube, too [207j At too short residence time, there is incomplete dissoziation, at too long residence time, there is dimerization At optimum gas flow, addition of 5% air to the carrier gas flow sharply loweres the shape of the signal because of consumption of H in the system to yield H,O 0 I % H, is necessary in the decomposition reaction at 800" The residence time of the gas In the cuvette is 0 1-0 5 sec [ 180,20S] Water vapour does not decline the sensitivity [206] Some authors recommend background correction with D, [62], others get accurate results without

3 2 2 2 3 Graphite furnace atomization of hydrides The sensitivity for Se in graphite furnaces is considerably lower than in flame-in-tube atomizers The reproducibility, however, is excellent The on-line atomisation approach utilises a direct transfer of hydride from the generator to a furnace at atomization temperature The generated hydrides are introduced into the in- ternal gas line of commercial furnaces To avoid contact with metal surfaces, however, hydride introduction through the injection hole of a graphite furnace via a sealed graphite tube is favourable [209] For the determination in excess of other hydrides (like As), the. HZSe was atomized from a pyrolytically coated tube a 2650" Because of interferences from volatile organics from the matrix, background correction was applied [2 101 Whereas a quartz- tube atomization device has to be constantly run at the atomization temperature, it is easy to use a graphite furnace for enrichment and pulse atomization The hydrides are trapped in a moderately heated graphite furnace, and subsequently atomized at high temperatures The trapping also eliminates effects of variable rates of hydride evolution Extraordinary rapid heating rates and thus high peak heights could be achieved at high heating rates, e g in a graphite- paper furnace [ 194,2091 Hydride trapping in a graphite furnace is greatly improved by addition of Pd to the furnace tube wall, which is added together with the surfactant Triton X-100, to get a homogenous distribution on the tube surface 121 I]

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3.2.2.3 Intermediate hydride trapping

For extreme trace analysis, enrichment of the hydrides in a cool- trap after generation in batch, and pulse atomization, together with simultaneous separation from H, and H,O vapour, greatly enhances the detection limit and lowers the background noise [6]. The time needed for the determination, however, increases at least 5-fold. Enrichment of H,Se also separates from excess H2 formed in the hydride generation reaction. Prior to cool trapping, the water has to be removed at first, because it clogs the cool trap and readily dissolves H,Se. Desiccants, like silicagel or Mg(CIO,), partially also adsorb H,Se [ 6 ] . CaCI, as desiccant also partially absorbs H,Se [ISI]. Therefore, the gases produced in the hydride generation vessel, are led through a cool trap with propanol/dry-ice or methanolldry-ice to freeze the water vapour, then the hydrides are caught in a cool trap with liquid nitrogen, and the excess hydrogen passes. The most difficult task is to separate CO, from H,Se, which is only essential, if glow-discharge atomization is used [212]. If a GC is placed between the stripping - trapping apparatus and the AAS detector, it is possible to discriminate methyl selenides from inorganic selenium, e.g. in the analysis of natural waters [ 18 11. Alternatively to purely freezing techniques, after passing a glass fibre filter for removing liquid droplets, the hydrides can be adsorbed from a stream of He on Chromosorb W in a vitreous silica tube inside a liquid N- cooled steel tube[6,213]. For desorption of H,Se, the cool trap is rapidly heated, either electrically or by immersion into hot water, leading to an atomization pulse. Alternatively, collection of the hydridem, - mixture in a high- pressure vessel, which is suddenly opened to the atomizer, also resulted in an enrichment effect [205].

3.2.3 DETERMINATION OF SELENIUM BY ELECTROTHERMAL ATOMIZATION

Matrix effects, spectral interferences, and volatility are major problems in the charring step. The analytical signal, however, is nearly independent of the speciation of Se. Full presence of matrix plus, spectral interference from Fe,and additional smoke from matrix modifiers necessitates Zeeman background compensation in many cases.

3.2.3.1. General atomization behaviour As analytical lines, 196.0 nm in the far UV, and the 4- times less sensitive line at 204 nm can be used. Optimized atomization temperatures found in the cited literature are medium, and vary from 2200 - 2700"; they depend on the tube material and the apparatus used. The differences between Se(IV) and Se(V1) - signals are within experimental error [63]. Similarly, no difference in the atomization signal between Se- methionine, selenite, and selenate at the 50 ng/ml level was observed [214]. Se-methionine in pure solutions, however, can be slightly lost even in the drying stage. For this reason, very slow drying of native serum samples is essential [215]. From 0.2% HNO,, in the absence of metal cations, losses from coated graphite tubes with platform occur even at 200°, but the presence of salts tends to stabilize the Se [63,216]. From acid solutions, Se may be also lost as H,Se [217].

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Perchloric acid in admixture with sulfuric acid severely suppresses the signal, whereas HCIOJHNO, mixtures yield higher signals than the individual acid [218]. Thus, acid contents and matrix have at least to be closely matched [ 1901. Mass spectrometric sampling of gaseous species that evolve from graphite include Se-oxides, carbides, hydroxides, the dimer, and finally the free atoms, as a function of temperature and oxygen partial pressure. In case of vaporization of pure selenious acid in dilute nitric acid from the graphite tube wall, the maximum of SeO' appears at 250°C, Se02' at 3OO0C, and the dimer at 700'C [214]. Beyond lOOO'C, free Se atoms is the sole species in the gas phase. Hydroxides are never observed in vacuum vaporization. Some metal ions, like Pd, severely influence this atomization behaviour (see later)[2 191. Se can also be atomized from a W- ribbon, which offers the advantage of very high heating rates (6000°/sec). As application, however, only the determination of Se in natural waters with Zeeman background compensation has been given [220]. The variation of the modulation frequency of the Se- hollow cathode lamps did not significantly affect the relative analytical performance, but there was significant variation in the limits of detection for different lamp geometries. A boosted hollow cathode lamp- configuration provides lower limits of detection [ 2 2 11.

3 2 3 2 Limits of deuterium background conipensation 3 2 3 2 1 General For ETA-AAS with D2- compensation, the maximum tolerable level for one atomization cycle was found to be 0 5 pg chloride, 4 pg sulphate, and 5 pg phosphate, otherwise the signal gets too low because of overconipensation Fluoride, however, does not interfere

If a proper separation procedure is appplied, the level of interferents is lowered, and deuterium background compensation becomes possible

[601

3.2.3.2.2 Interference of Fe In real samples, electrothermal AAS with a D,- compensation sytem is severely limited because of spectral interference of Fe. At 196.0 nm, there is a weak Fe- emission line within the spectral bandpass, leading to negative signals [222,223]. At high Se- levels, the line at 204 nm can be used, where there is no Fe interference, but spectral interference of Ni [223]. In case of overcorrection, standard addition gives too low results, because a part of the peak disappears in the overcompensated signal, but the added amount appears. If the atomization cycle can be timed very exactly, the slight delay of Se relative to Fe atomization offers the chance to cut away the wrong signals. Thus, a delay of 0.7 seconds made correct measurement in peak height mode possible. Mo- coating suppresses the negative overcompensation-peak up to 250ng Fe[2 141. Pd, Cu and Ni at elevated levels also produce over-compensation signals in H,SO,, but not in HNO, or HCI. This overcompensation can be overcome by charring with air at 300' [224]. Use of an extremely powerful "super-lamp" with significantly lower base- line noise made it possible to use large bandpass averages over a greater area by the background corrector, which increased the tolerance level of Fe [225].

426

3.2.3.2.3 Interference of phosphorus P on the level present in biological matrices causes overcompensation effects due to molecule absorption and stray light. Especially Ni and Pd, but also Ce, Pt, W, and Zr favour the formation of P-atoms and delay of the background, resulting in lower absorption of P- molecules and thus less base-line distortion [226,227]. In the analysis of serum in presence of C u M g - nitrate modifier, interference from P and Fe could be overcome by a delay of 0.7 sec in the read period, using a platform [228]. Similarly, in platform atomization, 5 pg P could be tolerated with D, in presence of 100 pg Ni. With Zeeman compensation, however, no effect from 10 pg P + 50 pg Ni was observed [229].

3.2.3.2.4 Applications done with D,- systems D2- compensation was sufficient for the analysis of river water in presence of Ni matrix modifier [230], in acetic acid extracts from plants with Cu as matrix modifier [63], or in marine samples with low levels of P [229]. At the low Fe levels encountered, wall atomization from pyrolytically coated graphite tubes was preferred over platform atomization [63] or uncoated tubes [231]. The normal range of [Fe] in serum is 0.5 - 1.5 pg/ml, which was already too much for coated tubes/ wall atomization/ Cu-Mg modifier [223]. After dilution of the sample, an albuminiPd - modifier in pyrolytically coated tubes delayed interferents sufficiently for D,- compensation [215], whereas other authors strongly recommend Zeeman compensation because of non- avoidable spectral Fe- interference [232].

3.2.3.3. Combination with liquid-liquid extraction After liquid-liquid extraction for separation from interferents, the organic phase can be directly injected into the graphite tube. Difficulties may arise due to undissociated evaporation of Se-chelates. Using dithiocarbamates or similar reagents, direct injection of the extract with diethyldithiocarbamate from acetate buffer pH4 into CCl, yielded best results [ 1 141. As the reagents are not completely destructed below 600°, a modifier to stabilize Se has to be added. Ni matrix modifier and H,O, as'ashing aid have to be added as aqueous solution prior to atomization. Pd as a modifier, however, can be added before the extraction procedure, because it is coextracted with the Se, like Cu, whereas Ni remains in the aqueous phase [224]. Gas stop in the charring step at 700" increased the reproducibility in non-coated tubes [233]. From liver digests, Se was extracted with ammonium pyrrolidine dithiocarbamate (=APDC) into CHCI,, injecting the organic layer into the furnace, preferable into pyrolytically coated tubes [234]. A graphite furnace was also used as detector of liquid chromatography of APDC complexes at C18- bonded silica gel columns. after elution with methanol, the organic phase was not used directly, but the fractions were evaporated, and redissolved with HNO,/Ni [86]. The extract of various piazselenoles in e.g. toluene can also be directly used for electrothermal atomization. The substituents on the piazselenole molecule influence the volatility of the undissociated species. Best results were achieved with the nitro- derivative, and worst with the unsubstituted [235].

427

Extraction of the 4-chloro- piazselenole into toluene and injection of the organic phase was applicated for the analysis of biological matrices [236] If addition of Ni- matrix modifier I S wanted, it has to added as aqueous solution into the tube, because i t is not coextracted

3.2.3 4. Stabilizing cations and matrix modifiers

3.2.3.4.1 General Losses of selenium from 0.2% HNO, solutions from coated graphite tubes with platform occur even at 200", as indicated with radiotracers. Se (VI) is less volatile than Se(I1) than Se(IV) [216]. Salts generally prevent volatilization of Se in the charring step Some of them act very effective (like Pd, Ni, Cu, Pt) and thus are added to various samples as matrix modifiers. The amount of stabilization, however, also depends on the tube surface and the kind of acid used. Additional smoke from the addition of these modifiers requires effective background compensation, which makes it necessary to use the Zeeman- effect, or Smith-Hieftje compensation. Rapid atomization from a platform inserted into the tube helps to atomize volatile elements before evaporation of the matrix, which is also applicable to atomization of Se [ 18,2281, In 0.2% HNO, and coated tubes with platform, even addition of 1% NaCl stabilizes all forms of Se in the charring step; selenate is completely stabilized up to 900". and selenite at 70-80% up to 800". If losses occur during the drying step, however, they cannot be prevented by salt addition, but only by very slow drying [216]. By means of 75-Se tracer, inorganic Se was quantitatively stabilized during charring from 3% HNO, solution on pyrolytically coated graphite tube walls by Sb, Cd, Mn, Mo. Ni, KJ, KJO,, Ag, Th, TI, Zn, and Zr. For charring of organoselenium compounds in urine under the same conditions, however, only Mo, Ni and Ag were effective. Contrary to other authors, Cu and Fe were found to stabilize inorganic Se only partially. Also, J als KJ ena- bled ashing up to 1000". In urine, KJ, Mn, Zn, Th, TI and Zr did not stabilize [237]. In a similar investigation using non-coated tubes and peak height evaluation, selenite in 5% HN0,- solution was stabilized during the charring step by Ni, Cu, Zr, Pt and Pd. To the contrary, Mg, Fe, Na, Al, Au, Ca, Mn, Cr were not effective [238].

3 2 3 4 2 P d Pd is partially maintained in the furnace up to 1700" [224] Mass spectrometric sampling of gaseous species that evolve in the atomization cycle from pyrolytically coated graphite tubes revealed, that Pd inhibits the Se-dimer formation in all cases it is just introduced, but not thermally pretreated Simul-taneously, Pd inhibits the formation of hydroxides and lowers the amount of vapourized monoxide SeO? is inhibited by Pd only, if Pd has been thermally pretreated It traps SeO, from the gas phase to yield a PdlSelO compound, which turns to be Se-Pd at 1 200", and atomizes as such [2 191 In simple Se(IV) solutions, Pd stabilized Se during charring in coated tubes t i l l 1200" P391 For the analysis of Se in serum, Pd modifier was pre-iiijected into the furnace, dried, and then the 111- diluted sample was added [239] Addition of 0 I % albumin together with Pd under certain circumstances doubled the signal of Se from serum samples in coated tubes The albumin was converted into graphite, proved by scanning electron microscopy Similarly, polyvinylalcohol was converted into graphite, which also improved Se- sensitivity in coated tubes to a lower extent 40 pglnil

428

Pd were found sufficient, and 150 pg/ml Pd caused the albumin to precipitate. At the 1.5% level of albumin, however, severe background absorptions were seen with D2 compensation [215]. In TaC- coated graphite tubes, peak heights and peak areas reached maximum values within the range of 1-10 pg Pd per atomization cycle. The TaC- coated tubes could not used without Pd, which yielded additional thermal stabilization. After extraction of Se with DDTC, 3 pg Pd were found to be optimum for charring of the DDTC- extract at 700" in the TaC- coated tubes [224]. For charring micro-amounts of biological material inside the tube, Mg- nitrate can be added as an oxidant, if it is possible to cope with the additional light scattering during the atomi- zation by means of the Zeeman- effect. In the analysis of blood and serum, a combination of PdMg - nitrate allows the use of higher pyrolysis temperatures, delaying the background of e.g. CaHPO, matrix [227]. After separation as the piazselenol and stripping into the aqueous phase, Ni and Pd were equally sensitive as matrix modifiers. Platform atomization yielded greater sensitivity and better reproducibility than wall atomization from pyrolytically coated tubes [240]

3.2.3.4.3 Ni Ni forms stable NiSe through the reaction between SeO, and Ni. NiSeO, is only stable to 300" in an 0,-free atmosphere, and NiO decomposes at 400". The presence of solid Ni is essential for Se- stabilization; in solid sampling, matrix modification with NiO was not effective. Glucose changed the nature of the surface and lead to low recovery of Se in spite of Ni presence. Ni as chloride was less effective as modifier because it was not reduced to metallic Ni fast enough, whereas NiNO, is easily reduceable by H2 also [217]. In 2.7% HNO,/ 0.1% Ni as matrix modifier, 1000 times excess As, Sb, and TI, 5000 fold excess Pb, 7500 fold excess B and 10000 fold excess Cd did not interfere [241], and D2- compensation was sufficient. In the analysis of digests of geological material, however, variable signal depression lead to wrong results of standard addition technique with deuterium background compensation [233]. Therefore, separation from Fe, P and other possible interferents was done by volatilization of the analyte in a stream of oxygen [241]. Zeeman background compensation enabled measurement of Se in digests of geological material by high- speed atomization from a platform in a pyrolytically coated tube, in presence of IOOpg Ni per atomization cycle. Evaluation via calibration graph as well as standard addition were possible [222]. In the analysis of biological matrices, 100 pg Ni strongly accelerate the atomization of phosphate. 10 pg P were easily compensated with Zeeman- effect, whereas with D2 as modifier, upper limit of P was I pg P per atomization cycle [229].

3.2.3.4.4 Ag The efficiency of Ag as a matrix modifier is about like Ni, but Ag is presumed to precipitate in biological matrices as the chloride. When 20 pg Ag or Ni were present, loss of Se did not occur at 1000" charring. The atomization was improved to 140% [242,243].

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3.2.3.4.5 Pt Pt in its elemental state is not volatilized up to 2400" [225]. Addition of Pt allows charring of Se up to IIOO", but it slightly delays the appearance of Se, and lowers the sensitivity, both with platform and with wall atomization. Significant Pt amounts stay in the tube and cover the surface with smll pellets of Pt- metal. As Pt does not stabilize organic compounds, it is often applied in admixture with Ni [226] ( 1 5 pgNi + 60-120 pg Pt) or Cu. Only Ni, Ag and Mo have been shown to reduce the volatility of organoselenium compounds. A pyrolytically coated tube with off the wall atomization gave better sensitivity than an uncoated tube and was less prone to complications from the unburnt residual carbon, which might arise in the platform [23 I ] . In the analysis of blood and other biological fluids, addition of Pt/Ni matrix modifier favours the formation of gaseous decomposition products of phosphate and changes the absorption-time- profile of Fe. Ni and Pt together favour the formation of P atoms, which can be measured at 213.6 nm [226]. In a Mo- coated furnace, the use of a Pt/Ni matrix modifier resulted in complete separation of the appearance time of the Fe- signal and the Se signal [214].

3 2 3 4 6 H g Mixtures of HgCI,/PdCI, as a modifier were investigated in pyrolytically coated tubes up to 0 10% Each of them improved the signal, but they were superior in admixture Whereas Hg alone accelerated the peak appearance, Pd had nearly no effect, and the mixture even delayed, because Hg favours the atomization rate At 0 10% modifier and 1 100" charring, Al, Fe, Ca, Mg, K, and Na did not interfere when present in usual concentrations in digests of fly- ash samples, with SMITH-HIEFTJE background compensation [244,245] Among the anions, the appearance temperature increased from SO, to NO, to CI

3 2 3 4 7 C u Cu as the nitrate has early been found to stabilize the charring of Se like Ni up to 1250"

Its effect upon the absorbance signal from Se is much more pronounced for wall atomization at the 10 ppm and 100 ppm level of Cu, but equal to platform atomization at 1000 ppm Cu in the peak height mode After combustion of plant materials in oxygen, Cu as matrix modifier in dilute acetic acid was proposed (10 pg per atomization cycle) Acetic acid improved the atomization signal at > IM concentrations, but significant differences in performance between different batches of tubes were noted [63]

~ 4 1 1

3.2.3.5. Tube material and tube design For wall atomization, pyrolytically coated graphite tubed are superior for the determination of Se. In the use of platforms, however, optimum conditions seem to depend on the kind of sample and the kind of apparatus used.

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Soaking with W- solution lead to a change of the surface structure because of carbide formation, but to no improvement for the determination of Se from dilute nitric acid solutions[ 24 I]. Coating of graphite tubes with Nb or Ta resulted in better reproducibility and accuracy, at equal sensitivity for Se in presence of 50 pg Ni [233]. TaC - coated graphite tubes lead to additional thermal stabilization of Se in presence of excess Pd, but also caused some memory effects [224]. In boron-nitride coated tubes, retention of Se in HNO, was possible up to 500", but losses of Se(IV) from 0.1M- HCI occurred to 20-30% within the range of 200-500". The boron- nitride -coating, however, was destroyed after 30-35 firings [216]. Coating with Mo was achieved by injecting 4 times 5 5 pI of 5 % Mo solution and heating to 2500". The Mo- coating significantly suppressed the interference of Fe resulting from overcompensation of the D,- signal, due to delay of the Se in the peak height mode, in presence of P t N matrix modifier [214].

3.2.3.6. Use of auxiliary gases Addition of 10% H, to the inert gas (Ar) reduced the peaks of organic molecules during graphite- furnace atomization (like pentane, hexane, methanol, ethanol, and chloroform; except aromates) and doubled the peaks of Se- containing organicals. Thus, D,- compensation was sufficient to use a graphite furnace as a detector in gas-chromatographic separation of volatile selenium- containing species collected from ambient air [246]. 5% H? in Ar was used to reduce the Pt- modifier to its elemental state, which made i t far more effective [225]. Ashing of biological samples with O? inside the graphite tube may be risky, because Se forms volatile oxides [247]. Charring of dithiocarbamate- extracts at 300" in air doubled the signal of Se in TaC- coated tubes, because of improved destruction of the reagent [224]. CO was used as an auxiliary gas during charring up to 1000" at a flow rate of 0. I llmin to remove O? from the furnace. This prevented the formation and subsequent atomization of iron oxide, which interfered in the background correction of the Se- signal with D2 [225].

3.2.3.7. Solid sampling and slurry atomization For Se, solid sample atomization is only reasonable since the introduction of powerful Zeeman-background compensation systems, and availability of sufficient homogenous solid standard material. As best and universal modifier, a mixture with graphite powder was recommended. For Se, however, few reference materials were available, and the detection limit was not sufficient in any case [248]. Introduction of micro-amounts of solid samples cannot only done rized about 60pg sample from a homogenized tablet of geological material, which was introduced into the tube together with the carrier gas [249]. For the analysis of Se in metal chips, about I mg of the alloy was inserted into the furnace and atomized. For Se, an atomization temperature from Ni-base alloys of 2600" was needed, and a detection limit of 0.2 pg/g for 1 mg sample could be reached only Standardization had to be made against doped alloys of similar composition [2SO], because the sample was not completely vapourized.

43 I

In solid sampling of liver homogenate, the Ni/Ag- matrix- modifier was added to the sample before freeze drying and homogenization, to ensure sufficient mixing For Img sample weight of liver, a detection limit of 10 ppb could be achieved [247] In slurry atomization, the solid sample is mixed with a fluid for reasons of dilution, and pipetted into the graphite tube. For analysis of coal and coal fly ash samples, a solid slurry was prepared by ultrasonic mixing. Calibration was done against similar NBS- standard materials. Atomization was done from a platform inserted in a standards grooved tube. The solid was diluted with slurry was 5% H N 0 3 / 0.04% Triton X-100. A Zeeman compensation device was essential. Se was more difficult to determine than As, Pb, and TI. At 1900°, severe matrix absorbance appeared, which necessitated to atomize as low as 1 8 5 0 O . Pd as modifier sharpened the peak, but did not improve the accuracy [25 I]. For the analysis of milk powder, 0.5 g sample was mixed with 3 ml H,O, and 200 pl of this slurry added to lml RhiMg- matrix modifier (0.4% Rh(NO,), + 0.25% Mg(N0,)2.6Hz0 + 0.4% HNO, + 3% Triton x-100). Charring was done from a platform, Mg- nitrate acted as ashing agent. Evaluation was done via peak area and standard addition [252].

3.3 ATOMLC EMISSION SPECTROSCOPIC METHODS

3.3 .1 INDUCTIVELY COUPLED PLASMA - DIRECT ASPIRATION

The main atomic line of Se at 196.026 nm has a rather high excitation and ionization energy and therefore tends to peak rather high in the plasma [253], nearly like an ionic line. The increase of observation height in the pure Ar plasma, however, lowers the signalibackground ratio, because of strongly increasing background Similarly, the signal/ background ratio is worse in the high power-range and upon addition of 5% N, to the plasma gas [254]. For Se, no improvement by means of switching the plasma excitation frequency from 50 MHz to 100 MHz has been achieved [255]. As far as spectral overlaps are concerned, the main emission line of Se at I96 026nm may be interfered by an Fe-line at 196.059nm, if the resolution of the spectrometer is insuffi- cient [ 2 5 61. Detection limits of Se emission in the ICP using direct aspiration, are within the range 10-70 ngiml [255,257] and thus insufficient for the analysis of natural waters and digests of biological material [258]. Direct coupling of the ICP to a liquid chromatographic system with a flow rate matched to the usual nebulizer uptake, lead to a detection limit of about 0.1 pg Se at the insensitive line at 203.985 nm [89].

3.3.2 DIRECT CURRENT PLASMA - DIRECT ASPIRATION

In the DCP, matrix effects upon the emission signal of Se at 196 026nm by ionization enhancement are negligible The signal was regained in presence of up to 15 g/l Na, and Ig/l AI, Ca, Fe, Mg [259] The precision, however, decreases significantly at lower concentration levels [259] The detection limit of about 0 25 pg/ml [260] is insufficient for environmental samples

432

3.3.3 HYDFUDE METHODS

The hydrides of interest, together with a constant amount of hydrogen, are produced in a continuous- flow system, and introduced into an analytical plasma without further nebulization. Evolution of a smooth stream of hydrogen without bumping and splashing is essential. Low spectral background and lack of nebulization losses improve the detection capability about two orders of magnitude, which is sufficient for many environmental samples. The multielement capability of the instrument is limited to the hydride-forming elements, only Se(IV) is monitored, and all interferences of hydride evolvation (see chapter 3.2, 3.8) have to be considered. The AES signal stabilized about 1 min after introduction of the sample to the hydride system [261]. The optimum range of acidity of the sample depends on the exact geometry of the device, and the carrier gas stream used. Starting from HNO, gave a slightly lower sensitivity compared to HC1 [262]. For the stabilization of the gas introduction into the plasma, in the simplest approach, the spray chamber of the conventional ICP- nebulizer was replaced by the mixing chamber of the hydride system [257]. Alternatively, the hydrides and hydrogen have been separated from the reaction mixture via a 2m or 5m long silicon rubber tube placed in the stream of Ar-plasma gas [263]. A long and narrow gas- liquid separator was filled with Pyrex beads, which provided smoother separation of liquid and gas [264]. If the hydrides are introduced into a DCP, half of the Ar flow is directed through the hydride generator to carry the evolved hydrides into the plasma, while the other half passes its normal way through the nebulizer system. The reaction gases (mainly hydrogen) are dried by passing CaCI,, and delayed in a suitable delay tube before nebulization, to provide a smooth gas stream. To obtain lower detectable concentrations, hydride evolution from a lOml sample batch is also possible, leading to a detection limit of 15 ng Se. Matrix Ni was successfully masked with phenanthroline [260,265].

3.3.4 OTHER EXCITATION TECHNIQUES

Excitation of electrically conducting solid samples in a DC-arc between crbon electrodes at 30 A suffer from the short wavelength of the Se- main emission line at 196.026 nm. A stream of argon shielding gas (7.5 l/min), long exposure time (65 sec) and optical grating of 1200 grooves/mm were necessary to obtain reasonable signals on the spectrographic plate. This method was used to determine Se in various sulfides after mixing with equal amounts of ultrapure graphite [266]. Analytical lines which can be measured without a vacuum are found in the visible region, but the energy required for their excitation is so high that they do not appear in arc and spark spectra. If the sample is dried upon an Al-cup, which is subsequently used as the target of a hollow cathode discharge of 240W, ionic lines at 444.62nm and 444.95nm appear, which can be easily detected spectrographically, with a detection limit of Ing/ml, achievable in human serum [267]. Contrary to other excitation sources, in a microwave induced plasma (MIP) within a flow of 1.1 I/min He, the Se- lines at 206.279nm and at 203.985nm emit signals as well, but applications for real matrices are not given [268]. Vaporization from a Ta- strip into an Ar-fed MIP allows to evaporate the solvent after solvent extraction, and subsequent pulsed vaporization of the analyte [ 161. After pressure

433

decomposition, the detection limit of the MIP, however, was insufficient for the determination of Se in fish and vegetables Molecular emission cavity analysis (MECA) uses the broad emission of SeO, Se2, and Se02 in the range 330-530 nm, with a maximum at 413 nm Molecular eniission is

achieved by injecting the sample into a steel cavity placed in an air-H1-N, flame, and cooled on the backside As many metal ions interfere, and H2Se is much more sensitively detected than Se- oxyanions, the sample is introduced after hydride formation For reasons of enrichment, the H2Se can be caught in a cool trap, and evolved by immersing the trap into hot water [269,270]

3.4 DETERMINATION OF SELENIUM BY MASS- SPECTROMETRIC METHODS

3.4.1 GENERAL

Five selenium isotopes are naturally abundant: Isotope Natural

abundance 74-Se 0.87 YO 76-Se 9.02 YO 78-Se 23.52 % 80-Se 49.82 YO 82-Se 9.19 YO Mass spectrometric techniques have the advantage of large multielement capabilities, relatively simple spectra, and offer the possibility of isotope ratio measurements and isotope dilution analysis.

for more details see 3.6.1

3.4.2 ICP - MS

A horizontally mounted ICP - torch serves as sample introduction device for a quadrupol mass spectrometer [271]. Since this technique is rather new (first papers published about 1980), only few applications for the analysis of Se in real matrices can be found in the present literature. In case of Se, isobaric interferences of neighbouring elements hardly occur. Arsenic is monoisotopic at 75 m/z, where there is no Se isotope, and bromine is hardly found as a cation [272]. In matrices with high chloride content, or in case chloride containing acids have been used for sample dissolution, polyatomic ions containing CI are encountered in the mass spectrum [273]. Additionally, Se coincides with background peaks of the Ar-plasma itself, emanating from various Ar and C1 isotopes. 76 Se 36Ar - 40Ar 77 Se 40Ar - 37CI 78 Se 38Ar - 40Ar 80 Se 40Ar - 40Ar Only 82 Se is free from such interferences 12721. Spiking the sample solution with 10% propan-2-01 or introduction of 3% N2 into the nebulizer gas flow reduces these polyatomic signals; maybe there is competitive formation of Arc', ArO' and ArN' [273].

434

In ICP-MS of positively charged ions, without any matrix, a detection limit of only 0.8 pg/l could be reached within 10 sec single ion monitoring, according to its high ionization potential of 9.75 eV [274,275]. In multielement analysis of natural lake waters, the detection limit of only 3pg/1 Se was insufficient ( 2 sec integration) [275]. In acetate buffer leaches of soils, monitoring of 78-Se and 82-Se resulted in a detection limit of 5 pgil in a 20 sec total measurement time. Noteably, the sampling for the mass spectrometer was done at 22 mm above coil, which is unusually high, but reasons were not given. However, the precision of the recovery of spikes to the acetate buffer was only 128% [276]. Direct use of non-digested urine and serum samples into the ICP-MS yields polyatomic fragments. At mass 82, where there is no Ar-Ar or Ar-CI background like for the other Se- isotopes, the results were anomalously high, in comparison with ICP-AES and AAS determinations. This may be due to the CCI" ion, which also appears in the spectrum of pure trichloro-acetic acid [277]. In the direct analysis of red blood cells, 74-Se coincided with FeO' [278]. In the negative ion scan, there are considerably fewer background peaks. The background peak at 78-Se disappears, but at 80-Se it is still present [279]. Introduction of hydrides into the ICP-torch reduces the number of interfering molecular ions in the mass spectrum, e.g. oxides and chlorides, but the start from Se(1V) and interferences in hydride generation have to be considered. A debubbler to remove excess air prior to the mixing zone greatly improves the results [278,280]. ICP-MS via the hydride was applicated in hunian metabolic studies employing stable isotope tracers [278].

3.4.3 OTHER MASS SPECTROMETRIC METHODS

In spark source mass spectrometry, the electrical conductivity of selenium in most of its compounds as well as in most of the matrices of interest is insufficient. It needs to be electrolytically preconcentrated at gold- electrodes after suitable sample decomposition by wet chemical methods, or reduced by means of hypophosphoric acid after addition of gold chloride spike. The resulting gold containing Se, Te and some other trace metals, is directly sparked. For quantitation, isotope dilution with 78-Se [45] or with 82-Se [281] has been applied. Thus, analysis of Se in coal, in steel and Ni- based alloys down to 0 1 pgig is reported. Both selenite and selenate evaporate as negatively charged ions from a hot metallic ribbon, which has been used to determine Se- traces in natural waters ("negative thermionic MS"). Selenite and selenate could be discriminated after separation by anion exchange. Within the range pH 1-12, no isotope exchange between selenite and selenate could be detected [282].

3.5 ELECTROCHEMICAL TECHNIQUES

3.5.1 PREFACE As selenium can occur in various oxidational states, For pure aqueous acids or alkalis, the normal potential of the respective redox reactions are [283]: 1M acid: I M alkali: Se'. <-0.92 V> Se ~ 0 . 3 7 V> SeO,'. <+0.05 V>SeO,'-

H,Se <-0.40 V> Se <+0.74 V> H,SeO, <+1.15V> Se0,'-

435

Common to all techniques for real samples (except for detection of compounds in HPLC) is to start from Se (IV) The formation of compounds with the electrode material on its surface can lead to shifts in the peaks obtained, as well result i n electrochemically inactive compounds Direct applications of electrochemical methods have been given only for saline waters, and for soil extracts obtained with neutral salts

3.5.2 DETERMINATION OF SELENITE IN AQUEOUS SOLUTIONS

3.5.2.1 Mercury electrodes

3.5.2. I . 1 Dropping mercury electrodes In 0.2M - HCI, the polarographic wave at -0.54V vs.SCE, deriving from the reduction of HgSe to Hg + H,Se, yielded a non- linear calibration graph Pb, Cu, and Fe influence both peak position and current, because of formation of selenides at the electrode surface [284] In 0.1M- HCIO, as well as in O.1M- KNO,, excess of Pb, Cd, and Cu decrease the Se- peak at -0.61 V vs. SCE, without change of its shape [285]. Even at pH=I, Pb can be masked with EDTA [286]. In weak acid ammonium sulfate, nitrate interferes, and has to be removed, eg by short warming the sample with ethanol [ 1291. In 1M NH,CI or NH,- acetate soil extracts, in'the range pH 5 4 - 9.0, an analytically useful peak could be obtained within the range - 1 . I V to - I .6V vs SCE. The best separation of the selenium current peak from that of the supporting electrolyte was obtained at pH 8. with peak potential of -1.34V versus SCE, and a detection limit of 5ng/ml. In this buffer solution, Fe, Pb, and Cu do not affect in 1000-fold excess, and Co, V, Te, Cr(lIl) , Mo do not affect in 100 fold excess over Se. The interference from Zn is removed by addition of EDTA, which simultaneously causes e peak from the reduction of Pb-EDTA at - I .2V, but in real samples the Pb- concentration is usually too low to interfere Among the organics, at the potential and pH conditions used for the reduction, the most likely interferents are expected to be simple organic compounds containing carbonyl and carboxyl groups Only maleic acid was found to interfere, and could easily be removed by mild acid hydrolysis [284]. In acetate/borate/phosphate buffer pH 4.0, the second peak of the Se- reduction at - I . 3 I V vs. SCE could be used in presence of large excess of Cu, Pb, and Cd [285]. In order to avoid precipitation of selenides, formation of selenosulfate from selenite was achieved by adding Na-sulfite to the acidified sample solution, with subsequent adjusting to pH 7-8 [ 129,2851. At pH 9-1 1 in ammonium sulfite solution, Se can be measured by reduction of selenosulfate at -0.95 V down to 5 ngiinl [129].

3 5 2 1 2 Stationary Hg - drop electrode

In a first step, Se is deposited and enriched by electrolytical reduction at the working electrode Cyclic voltammograms show, that a solid HgSe - film develops on the drop surface, which dissolves at a more cathodic polarization [287] In the presence of halide ions, the cyclic curve is modified, and the wave shifted to more negative values From selenous solutions containing halide ions, a selenium compound is deposited on the surface of the Hg drop, which transfers no electricity, but is cathodically reducible, termed HgSe

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The observed shift in the peak potential is due to complex formation [287]. At increasing plating time, an additional peak at more negative potential appears in cathodic stripping, which is less at lower pH, and does not occur at pHc4.2. The sum of currents of the two peaks is constant, and equal to the intensity of the peak when only one is present. This is interpreted by the formation of a mixed deposit of Hg-Se", and the formation of Se" by reaction between H,SeO, and H,Se [288]. In presence of Cu, a selenide presumably containing Cu(1) is formed during the electroplating step, instead of the Hg-Se compound [289].

Anodic stripping voltammetry The position of the anodic stripping peak strongly depends on the pH of the solution. The determination is interfered from many elements deposited together with Se, and also from compound formation at the electrode surface [290]. In the analysis of milk powder, a matrix very low with respect to trace metals, the direct anodic stripping voltammogram of a decomposition solution with HCIO, yielded high background currents, which could be reduced by fuming with HCIO, [291]. Increased sensitivity could be reached by scanning in the differential pulse mode, with an amplitude o f f 20 to 50 mV.

Cathodic stripping voltammetry Besides As, Se, and Te, also V, Cr(VI), Mo and W have been determined by this technique [292]. Cyclic voltammetric studies at a stationary hanging mercury drop electrode in acetate buffer in the range pH 3.85 - 5.55 shows three electrode reactions. At -0.3 V vs SCE, a broad and completely irreversible peak appears, which is due to the reduction of selenite to elemental selenium, and which is pH dependent and temperature - dependent. In cathodic stripping voltammetry, the two other peaks, at -0.75 V and at -0.90 V are utilizable. The third peak at -0.90 V is not observed at pH 4.2/30 sec plating time, but increases with increasing plating time. This is explained by both the formation of a mixed deposit og Hg-Se", and the formation of Se" by reaction of selenite with hydrogen selenide [288]. The selenium peak in the cathodic stripping voltammogram is shifted with pH, beause of participation of protons on the reduction to H,Se. The dissolution current is proportional to the electrode surface, which indicates, that the reduction takes only place at the electrode surface [293]. Increase of pH reduces the peak current, and shifts the dissolution peak to more negative values. At pH '8, no cathodic dissolution peak was observed [294]. The cathodic stripping voltammetry of Se in 0. IM HCI yielded a non- linear calibration graph for to 10"M Se. Variation of the deposition potential in the range of +0.05 to -0.30 V VS. SCE was of low influence [295]. The presence of metal ions may shift the stripping peak to a more negative potential. The determination of Se is interfered by Pb and Cd, which strongly suppress the peak at 1 mg/l already. The suppression is less in presence of some Cu, when deposition at -0.3V is used [36]. Zn and Cd make no peak themselves, but they severely suppress the Se- signal [294]. In dilute HNO,, 20 - 100 fold excess of selenide forming cations can be tolerated, but Pb and As interfere [296]. Arsenic forms a new peak at -0.3V vs. Ag/AgCl along with Se. In ammonium sulfate at pH 4.5, addition of Cu (up to 1 pg/ml) enhances the deposition of Se at Hg, and shifts the peak potential to more negative values. During cathodic

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stripping, presumably a Cu-Se compound is dissolved within the range -0.8 1 to -0.84 V. Like Cu, also Bi, Ag, and Au shift the stripping peak potential to more negative values, with respect to the Hg-Se peak, which leads to peak enhancement and to peak splitting at deposition potentials more positive than -0.5 V. From the position of the peak potentials it can be concluded, that Cu-Se is the most stable compound investigated [290]. Whereas the cathodic dissolution peak of selenium in dilute H,SO, and H,PO, is found at -0.51 V vs. SCE, it moves to -0.704V in IM- ammonium sulfate/0.4M- EDTA/pH 4, and to -0.81 V in 0.3M KNa- tartrate/0.2M-EDTA/pH 6 [294]. Interference from Cd, Zn, As, Cr, Pb and W was minimized by utilizing ammonium sulfate/EDTA/pH 4 as supporting electrolyte. Cu, Sb, Ti and TI in excess still interfere. The surfactant Triton X-100 at > 1 O-'% completely suppresses the peak [294,295]. Cathodic stripping voltammetry in ammonium sulfate /EDTA at pH 2.3 in presence of 2 pg/ml Cu, enables the specific determination of selenium and tellurium in presence of large excess of each other [293]. Cu concentrations of 3 pg/l or more enhance the height of the cathodic stripping peak. In presence of Cu, peak height and peak potential largely depend on the deposition potential At deposition potentials more positive than the formation of Cu?Se, the stripping peak shifts to more positive potentials, and the peak height decreases [289,297]. Cathodic stripping of Cu-Se yields a narrower and higher peak than for Hg-Se. Optimum sensitivity has been achieved at pH 1.6 and 40 pM Cu [289]. In dilute HCI, Fe, Pb, Zn, Cd, and Te decrease the cathodic stripping peak; Zn and Pb can be masked with EDTA. After deposition at -0.35V vs.Ag/AgCI, the cathodic stripping peak is obtained by sweeping the potential of the working electrode to -0.9 Vwith a speed of 10 mV/sec [297]. Addition of Cd caused a decrease of the stripping peak of Cu,Se, while 2 new peaks were formed, due to Cd reduction, and the cathodic dissolution of CdSe. Cd can be partially masked with EDTA [297]. Cathodic stripping voltammetry at the hanging mercury drop electrode can be directly applicated to the analysis of drinking water. Ammonium sulfate as the supporting electrolyte, as well as EDTA to mask interfering cations, are added to the sample, the pH adjusted with sulfuric acid to pH 2.2, and 0.1 mg/l Cu to improve sensitivity. After 5 min plating at -0 .254 vs Ag/AgCI, a detection limit of 1 pg/l at the stripping peak at -0.68 V could be reached. Sulphide strongly interferes [298]. For the determination of Se(IV) in sea water, only 1/1 HCI and Cu have to be added to adjust to pH 1.6. After 15 min of deposition at -0.4 V, down to 0 7 ng/l Se could be detected [289]. After volatilization of Se from most of the matrix in a stream of oxyge, and dissolution from the cool finger with dilute HCI, Se was determined by cathodic stripping voltammetry with ammonium sulfate/EDTA pH 4.5 as supporting electrolyte, and addition of some Cu [621. Direct use of the digestion solution from biological materials (liver, rapeseeds) resulted in no peaks in cathodic stripping voltammetry because of high background. Separation via extraction of a piazselenol with subsequent wet ashing of the extract lead to suitable sample solutions ready for the determination. Without the evaporation of the organic solvent, however, resulted in sensitivities less than 30 % with respect to the aqueous sample [26]. For the determination of selenium in biological materials, Se was separated and enriched from the acid digest by sorption upon an anion- exchange resin in the acetate form at pH 3, prior to either cathodic stripping or anodic stripping voltammetry [36].

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3.5.2.2 Au- electrodes

In anodic stripping voltammetry of selenium from a Au-disk electrode in 1M- H,SO,, stripping peaks appear at +0.64V, +0.86V, and at -1.03 V. Hg interferes [299]. Similarly, in 0. IM-HCIO,, three anodic stripping peaks are observed for large quantities of deposited Se. At the beginning, and at low concentrations, approximately a monolayer is deposited. Thus, the anodic stripping of very small quantities of Se following deposition at a low flux yields a single anodic peak at 0.8V. Further electrolytic reduction of Se leads to irreversible diffusional transport of Se into the electrode, forming a Au-Se alloy of unknown stoichiometry [300]. In the anodic stripping voltammogram following deposition at high fluxes of Se on the Au- surface, two additional peaks, at +0.63V and at + l . 15V are obtained [300]. These peaks can be interpreted as due to bulk Se, adsorbed Se, and as intermetallic Au-Se compound of unknown stoichiometry. Only the adsorbed amount of Se is analytically usefull. To enable only formation of adsorbed Se upon the electrode surface, a deposition potential of 0.15V vs. Ag/AgCI is preferable over the deposition at more negative values. The optimum deposition potential at the Au-electrode is thus far more positive than the -0.35 V for the mercury drop electrode for the same sample solutions. This also reduces the extent of codeposition of interferents, such as Cu and Pb [36]. The Au- electrode was pretreated prior to each experiment by polishing the surface, and preconditioned in 0.2M- HCIO, by applying alternate cycles. However, difficulties in obtaining reproducible Au electrode surface area from one experiment to the other arose. The anodic stripping procedure at the rotating gold electrode equalled the sensitivity of the cathodic stripping procedure at the mercury electrode, but the reproducibility was worse [36]. To make the Au- surface continuously renewable, the Au can be plated upon glassy carbon prior to each run. Detection limits were 4 times less than for pure Au electrodes, but only I@'M Au was needed in solution, which made it rather cheap [301].

3.5.2.3 Graphite and carbon electrodes At a graphite-pin-electrode, made a mixture of graphite and polyethylene- powder, the reduction of selenite gives two polarographic waves, within the range 0 to -0.2 V VS. SCE, and at -0.6 to -0.8V , which correspond to the reduction of selenite to elemental selenium, and of elemental selenium to selenide. At electrolysis in the range of the more negative maximum, the electrode surface is partially covered with red selenium, another part precipitates as colloid near the electrode in the solution. This amorphous Se is electrochemically incative. Its relative amount incrreases with pH, and is at maximum at a plating potential of -0.3 to -0.4V [302]. Increasing acid concentration increases both the anodic and the cathodic stripping peak of elemental hexagonal metallic selenium. During electrolysis in concentrated HCI, however, elemental chlorine formed at the counter electrode interferes by oxidation of selenite to electrochemically inactive selenate [302].

Anodic stripping voltammetry At a graphite disk, impregnated with wax polyethylene = 3 I , after electrolysis at -0 6 V vs SCE, anodic stripping voltametry can be performed in dilute HCI, which results in a peak at +O 22V for selenium Cu is added to increase the electroactivity of Se deposits because of formation of an intermetallic compound, but produces a second peak at -0 15V To obtain maximum sensitivity, Cu Se must exceed 30 1 [303] Acidic electrolytes give much better sensitivity for Se than neutral or basic ones After 1 min depositlon time a

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detection limit of 0 1 pg/l was achieved for pure solutions [304] Upon the addition of so- me Cu, the peak of selenite ion increases and moves towards a more positive potential, which suggests the formation of an intermetallic compound At large excess of Cu, the anodic stripping peak for Cu appears at about 0 (vs AgIAgCI), which overlaps the Se- peak [304] Matrix silver delivers a peak at +O IZV, and has to be removed beforehand, whereas Pb increases the Cu-Se signal, and Fe and Mn are of no influence [303]

Cathodic stripping voltammetry: At a graphitelwax electrode plated with Hg, contrary to the Hg drop-electrode, it is possible to determine Se by cathodic stripping in acid as well as also in alkaline sample solutions (> IM- NaOH), which masks Cu and other metals The sensitivity of the cathodic stripping peak is effected by the deposition potential and the time, the scan rate, and the thickness of the Hg- film. As optimum conditions, plating at -0.10 to -0.30 V, and potential sweep of 60 mV/s has been found [305]. For cathodic stripping voltammetry upon a rotating graphite disk electrode, Se is plated for 1-5 min in 0.4M H,SO,, in presence of Cu and chromate, and subsequently stripped in the pulse mode, to obtain a peak at -1.07V The method was applicated to the determination of Se in matrix Ga, after separation from the matrix and from nitrate, deriving from the dissolution procedure [306].

3.5.3 DETERMINATION OF PIAZSELENOLS

Isolation of selenium as a piazselenol, and the subsequent polarographic determination of the piazselenol itself enables separation and enrichment from interfering matrices (see 2 1 1 , 2 3 4 , 3 1 1 ) In 0 IM NH,CIO, i n formiate buffer at pH 2 5 , polarographic waves of the piazselenol from 3,3'diaminobenzidine at -0 1 1 V and at -0 63 V vs SCE are obtained at the dropping Hg- electrode The reagent itself gives reduction peaks at -0 4 I V and -0 97V A detection limit of 0 4 ngIml could be obtained [307] Similarly, at the hanging mercury drop electrode, adsorptive stripping voltammetry of the complex of Se with 3,3'dianiinobenzidiile in the differential pulse mode enabled the detection down to 0 2 pg/l [295] For the analysis of effluents from the mining industry, Se is extracted with o-phenylenediamine from ammonium perchlorate buffer pH=9 with toluene After addition of acetone and NH4CI/HCI - buffer pH1 3 , a homogenous solution with sufficient electrolytical conductivity is achieved to enable the polarography of the extract with a dropping Hg electrode Under conditions of anodic polarization, only one polarographic wave of Se is achieved, which yields niaximuin current at pH I 3 , leading to a detection limit of lygll in the original solution Prioi to the extraction, Cu was removed by extraction with dithizone, and large excess of Fe was masked with EDTA [308]

3.5.4 ELECTROCHEMICAL DETECTORS IN LIQUID CHROMATOGRAPHY

Liquid chromatographical separation on a cation exchange resin with on-line detection of Se at a tubular Au- electrode helps to cope with interferences of codeposited metals in anodic stripping voltammetry When Se is eluted from the column, the potential I S set to -0 30V, and the reduction current is monitored The electrode surface IS cleaned by rapid

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cycling between -0.30 and +1.20 V, and kept inert during regeneration of the ion-exchange column at +1.30 V [92]. Selenols, diselenides, and selenenyl sulfides could be detected by group- selective electrochemical reactions after reversed phase liquid chromatography at two dual Hg/Au amalgam electrodes in series in the eluent stream. The upstream electrode is set at - 1.1OV VS. Ag/AgCI to reduce disulfides, diselenides and seleny sulfides, or at -0.5SV to reduce only the Se- compounds. The selenols are detected directly by facilitation of the oxidation of Hg from the downstream electrode, when it is set to +O. 15 V vs.Ag/AgCI. Diselenides and selenenyl sulfides are determined by first reducing them to the selenol and/or thio form at the upstream electrode followed by detection of the selenol and/or thiol at the downstream electrode [90].

3.6 RADIOCHEMICAL AND NUCLEAR METHODS

3.6.1 GENERAL

As can be seen from table 1 , activation with neutrons leads to isotopes with overall soft b-lines. For their detection, the NaJ(TI)- detector is up to 10 times more sensitive, but lack of selectivity requires chemical separation procedures. With the Ge(Li)- detector, direct counting after activation is possible in some cases [9].

--Tables 1 and 2

3.6.2 ACTIVATION WITH THERMAL NEUTRONS AND PURELY INSTRUMENTAL

DETECTION OF 75-SE

After sealing in polyethylene or quartz vials, 30h irradiation with thermal neutrons and 2-3 weeks storage, 75-Se could be determined by y- counting at 265 keV with a Ge(Li) detector. In particulate matter from riverine and marine waters [3 171, in atmospheric particulates [ 145,3181, coal, fuel oil and fly-ash [319], detection limits of 25 ng abs. [313] resp. 10 ng/g [145,3 181 were achieved. For biological materials, like tissues, blood plasma, serum and erythrocytes, even 5 days of irradiation, 6 weeks of cooling, and half an hour counting time with a Ge(Li)- detector were necessary [3 19,320,3211. At 264.6 kEv, 182-Ta at 264.6 keV cannot be discriminated from selenium because of its quite similar half- life [3 131, but this is not crucial for biological matrices.

3.6.3 ACTIVATION WITH EPITHERMAL NEUTRONS

During activation with epithermal neutrons only, contrary to interfering concomitant elements, the cross section of 74-Se to yield the y- radiating isotope 75-Se, is not entirely lowered, which improves selectivity [32 I ] . The production of 24-Na with epithermal neutrons is much smaller than with thermal ones, and the overall activity is about 20-fold less after Cd shielding [323]. Similarly, in epithermal neutron activation of silicate rocks and sediments, the interference of 181-Hf and 131-Ba at the 75-Se line at 136 keV is

44 I

lowered, when epithermal neutrons are used [312]. However, at the 136 keV- line, the radiation from 99m-Tc at 140 keV, which is a daughter of Mo, is of equal sensitivity to the radiation of 75-Se at 136 keV [315]. The line at 265 keV is not observed in any sample because of insufficient detection limit [3 121. For the analysis of selenium in coal and fly ash, epithermal irradiation was found preferable, because of better precision, and equal sensitivity with respect to conventional y- irradiation method. Ash samples had to be irradiated for 1 day, and coal samples for 2 days. After 20 days of decay time, the 75-Se could be counted at 265 keV, achieving a detection limit of 0.1 pg/g [314]. In case of biological matrices, the improvement of using epithermal neutrons with respect to thermal ones was found to be negligible [321].

3.6.4 METHODS OF ACTIVATION AND SUBSEQUENT CHEMICAL SEPARATION

For counting the low-energy y- emission of 75-Se, chemical separation from interfering matrix elements allows to use the less selective, but more sensitive scintillation detector Addition of up to 1 OOmg inactive Se- carrier allows to do the clean-up on a macro- scale, and to control the regain of the analyte After coprecipitation or adsorption, active Se can easily be counted on the solid phase

3.6.4.1 Geological materials

From acid digests of geological materials, Se was separated and counted on the solid after precipitation with sulfite [51,54,3 16,3241, with thioacetamide [325], with MnO, [IS], or sorbed on A&O, [326]. Interfering activated Ta was removed by coprecipitation with inactive Ta carrier from acid solution [326]. Alternatively, distillation as the bromide [54,57] as well as various extraction methods were utilized with activated samples [ 54,325,3271. Decomposition with alkaline fluxes after irradiation and addition of Se- carrier quantitatively yields soluble selenate, which is not precipitated along with hydroxides (Fe) or sulfides in alkaline sodium sulfide solution [325]. For large sample weights, Se and Te were extracted together with A u and Ag by fire assay in a flux consisting of lead oxide, soda, quartz, sugar, and borax, into newly formed metal- lic lead. Only this lead button was activated. The active material in matrix Pb is less hazardous to handle. However, 203-Pb emitted at 279 keV close to Se, and had to separated prior to counting. After dissolution in nitric acid, Se was reduced to the elemental state with hydroxylamine, and counted on filter [328,329].

3.6.4.2 Biological samples

After irradiation with thermal neutrons and 1-2 weeks of cooling, inactive carrier I S added If the samples are burnt in a stream of oxygen, Hg and Br come along with Se, which may interfere in radioactive counting This has to be also considered after distillation as bromide [309,333] Se was separated from Hg and Br by solvent extraction [327,330], adsorption on charcoal [64], or 82-Br was just allowed to decay within 20 days [I21 From acid digests of activated biological samples, Se was separated by liquid-liquid extraction as piazselenol [9,105], as carbamate [327], or as iodide into CC14 [53] Direct counting was possible after sorption as the dithizonate on carbon powder at pH=8, or after coprecipitation with Fe- hydroxide in presence of ascorbic acid [332]

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3.6.4.3 Water samples

Water samples were reduced with ascorbic acid at pH=2, and Se was adsorbed on active charcoal, which was finally activated and counted [ 1361.

3.6.5 USE OF THE SHORT-LIVED 77M-SE

76-Se can be activated to yield the short-lived 77m-Se with a half-life of only 17.5 seconds. No separations are possible, but nearly no handling is necessary. 28-AI, together with Ti, Mg, V, Mn and U, is also accessible to short time activation, decaying with a half-life of 2.3 min at 1779 keV [334]. In geological and coal matrices, short-lived Se can be interfered with 28-AI, causing high dead times and high background [313]; therefore, 77m-Se activation has been mainly used for biological and related matrices. Short activation of 77m-Se minimizes matrix activation. The main sources of error are the variation of the neutron flux [335], and exact timing. Biological samples were simultaneously treated with aqueous standards in cycles of 20 sec irradiation, 3 sec waiting, and 18 sec counting at 162 keV with a Ge(Li) detector The delay of 3 sec between irradiation and counting periods was necessary to decay 38m-CI with its half-life of 0.7 sec [336]. Food samples were irradiated for 20 sec, decayed for 20 sec, and counted for 20 sec at I62 keV for 77m-Se. The precision significantly improved by recycling the samples up to 4 times [337,338]. 200 mg of biological material was irradiated for only 2-4 seconds in a high flux of 10'' n/cm2.s, and measured I 5 sec after the end of the bombardment at 162 keV, thus achieving a detection limit of 5ng/g [335]. Similarly, for determination of Se in liver tissue, samples and liver reference standards were irradiated separately and in fixed sequence for 90 sec. The integrated value of the current neutron flux was determined by use of gold monitor samples [339]. In atmospheric particulates, a detection limit of I8 ng Se and a precision of 13% could be achieved after 60 sec irradiation with 5.10'' n/cm'.s, 5 sec delay and 30 sec counting [3 131. Alternatively to thermal neutrons, the 77m-Se could be also produced by 2 min photon activation from a 5-10 kCi co-60 source, but the detection limit of 1 mg Se in a pellet of 20g of animal food stuff was rather poor [340].

3.6.6 PHOTON ACTIVATION

With high energy photons of about 35 MeV , two nuclear reactions with Se- isotopes can occur [341,342,343,344]: 76-Se (y,n) 75-Se 120 d 265 keV/ 136 keV 82-Se (y,n) 8lm-Se 57.3 m 103 keV High energy photon activation has been applicated to determine Se in the multi-element analysis of river sediments, atmospheric particulates, and related materials. The samples were pelletized with Li,SO,, encapsulated in Al for neutron capture, and simultaneously irradiated with standards.After irradiation, the Al-foil had to be discarded because of to reduce background from 27-Al(n,y)24-Na [344]. Similarly, atmospheric particulates on polystyrene filters were pelletized along with elemental and flux monitors, and submitted to multielement photon activation with Ge(Li) detection [345]. Soil and fly-ash samples were irradiated, cooled 1 day, pelletized with cellulose powder, and finally measured with a planar intrinsic Ge-diode [343]. At 136 keV,

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the limit of practical determination was estimated to be 0 2 pg/g, there was some interference by 57-co

3.7 X-RAY - SPECTROMETRIC METHODS FOR T H E DETERMINATION O F SELENIUM

3.7.1 GENERAL

For analytical purposes of Se, the K a - emission lines are exclusively used. They hardly coincide with neighbouring elements, or with L - lines of heavy elements (see table 3).

Table 3 Wavelength and energy of some X-ray lines [346] Element line wavelength energy keV As Ka2KL2 1 1799 10 508

K a l K L 3 11759 10 544 Pb LalL3M5 1 1750 10 552 Bi La2L3M4 I 1554 10 731

LalM3L5 1 1439 10 839 Ge KR3KM2 1 1294 10 978

KBlKM3 11289 10 982 Se Ka2KL2 1 1088 I I 181

KaIKL3 1 1048 I I 2 2 2 As Kfi3KM2 I0578 I 1 720

KDlKM3 I0573 I I 726 Br Ka2KL2 10438 1 1 878

K a I K L 3 10397 I1 924

In the wavelength dispersive mode, an overlap between the Se KOL2,3 line, the second order of the Hg LlM2 and the Hg L2M4 line [347] is reported.

3.7.2 WAVELENGTH - DISPERSIVE XRF

The best efficiency of Se-Ka line excitation is obtained with a Mo-target tube, which I S

used in most cited cases, but continuous background of bremsstrahlung may be high, thus decreasing the accuracy Excitation with a Ag- target tube yielded nearly the same power of detection, but with much less background radiation [348] As a compromise for the determination of 12 elements in pharmaceuticals after coprecipitation enrichment, a Cr-tube has been used [147], which allows to detect Se by a factor of 2-3 less with respect to a Mo-tube The excitation spectrum is usually discriminated with a 100-LiF- crystal, at an angle 26 = 31 87" After excitation with a Mo-target tube, backgiound correction on both sides of the Se-peak was done at 3 I 10/32 60°[ 1441, or at 3 I 0132 74"[349] In case less than 10 mg of solid per cm' are available for final determination, a correction for non-infinite sample depth has to be applied [350] For water samples, Se can be collected by coprecipitation and sorption methods, and the resulting uniform solid finally counted on the membrane filter As collectors, ZnS or CdS freshly prepared on a membrane filter, were successfull at pH 3-4 Coprecipitation with

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subsequent XRF- measurement has been done with polyvinylpyrrolidon/ thionalide at pH 4 [152], with diethyldithiocarbamate at pH 4.7-5.0 [150], or with combined diben- zylammonium and Na- dibenzyldithiocarbamates at pH 2.5 - 8.0 [ 1461. Similarly, from acid digests of biological materials and pharmazeutical preparations, Se was coprecipitated with the dibenzylaminosalt of dibenzyldithiocarbamic acid at pH 4 *I [147], or with Te [348,349]. Nitric acid interferes with the Te- reduction, and has to be reduced beforehand. Diethyldithio-carbamate as precipitating agent is not suitable in presence of large excess of Fe, which is also precipitated and can dominate the spectrum [349]. For the selective XRF- measurement of Se together with As and Sb, the corresponding hydrides were evolved from hydrochloric acid samples, and adsorbed on filter paper impregnated with AgNO,, which could be directly submitted to XRF measurement [347].

3.7.3 ENERGY - DISPERSIVE XRF

For Se, the line at 11.21 keV is used throughout, after Mo-K- excitation. Most authors prefer peak area evaluation, and background correction. Sample preparation procedures are not entirely different to wavelength dispersive methods. In soils and atmospheric particulates without chemical pretreatment, however, the detection limit of about 3 pg/g is not sufficient [350]. To reach the common geochemical level, wet- chemical separation from the matrix and reductive coprecipitation with Te has been proposed [ 1281. Se in serum and whole blood could be directly counted after drying a 0.75 pI sample on a filter spot. The peak intensities were corrected according to the intensity of the back-scattered Mo K X-rays in the range 14-19 keV. As detection limit, 60 ng/ml in 100 sec was reached (normal value: 80-120 ng/m1)[351]. For the determination of Se in water samples, elementary Se was adsorbed on active carbon after reduction of selenite with ascorbic acid. Both Selenate and selenite were reduced by refluxing with thiourea in sulfuric acid. For excitation of Se in the active carbon, a W-target tube was used, and the detection limit could be extended to SO ng/l by an extraordinary long counting time [134].

3.7.4 X-RAY SPECTROMETRY IN TOTAL-REFLECTION GEOMETRY

Total reflection of the incident beam yields excitation only at the sample surface [353,358]. Aerosol samples could be excited directly in the total reflection geometry, but digestion or low-temperature plasma ashing was preferred to obtain a more representativ sample [359]. For serum, 30 pl samples were pipetted onto a Si X-ray mirror, spiked with Ge as internal standard, and air dried. A W- target tube was used to excite a Ni- secondary target, yielding a narrow band-pass of primary X-rays (K-edge at 12.66 KeV), to excite selective Se at 13.0 and 13.3 keV, while at the same time preventing the excitation of a relatively high concentration of Br (K edge at 13.475 keV) in the serum [358].

3.7.5 PROTON - INDUCED X-RAY EMISSION (PIXE)

The determination of Se by PIXE has only been reported for biological matrices yet. If biological specimen directly interact with the proton beam, they should be placed on pure backing thin targets backed with nuclear graphite, to avoid problems with the stability of

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the shape of the sample. When the proton current exceeds 200 nA, evaporation of Se, Ca, As and K occurs [352]. The depth of penetration of exciting protons into the sample decreases with increasing average atom number of the sample, and ranges from 0.5 - 50 pm [353]. To reduce intense low-energy radiation, an Al- absorber of 78 pm thickness was placed in front of the detector [354]. By direct excitation with protons of 1.7 MeV, 0.08 pg/g Se could be detected [352] in a biological matrix Blood serum was dry ashed at 60°, ground, mixed with 20% graphite powder and Pd as internal standard, and formed to a thermally stable pellet. As the detection limit, 10 ng/g was found for 100 min counting after excitation with 1.8 MeV protons, or after 30 min counts after excitation with 4-MeV protons [ 3 5 5 ] . Alternatively, freeze- dried tissue and plant samples were converted into a fine powder, doped with Ag as internal standard, and fixed at the target frame by means of 1% solution of polystyrene in benzene [356]. From acid digests of biological matrices, Se can be selectively separated by coprecipitation with Te, and excited with 1.8 MeV protons, which leads to a detection limit of about 3 ng Se [42,354,357].

3.8 CONVERSION TO SE(IV)

In potable waters as well as after oxidational digestion, a substantial part of Se is present as selenate, which is advantageous because i t is far less volatile and adsorbable on solids. Prior to most methods involving chemical reactions, Se has to be converted to the quadrivalent form. Besides the conversion reaction, excess nitrous oxides and chlorine have to be driven off, which interfere with subsequent hydride formation, colour reactions, reductive precipitation, electrochemical reactions etc.. In addition to interferents present in the sample, incomplete recovery is possible due to incomplete reduction of Se(VI), further reduction to Se", or volatilization. Losses due to volatilization or precipitation at vessel walls can be traced with 75- Se labelled compounds Most authors use reduction reaction of selenate with hydrochloric acid: H,SeO, + 2 HCI = H,SeO, + H,O + CI, Different optimum conditions found in the literature depend on the acid mixture used, the amount of sample to be oxidized, as well as on residual organics and cations (Fe) in the sample (see table 4). Remaining nitrite, which may interfere further, can be destroyed by addition of hydroxylamine, sulfanilamide,or amido-sulphuric acid [ 18 1,360,3611. Dry- ashing with Mg(NO,), and dissolution with 6M-HCI on the boiling water bath yields selenite and complete oxidation of organics, which renders a conversion step unnecessary. Besides heating with HCI, conversion was also achieved by UV- irradiation at pH> 7.5 W I

446

Table 4. Quantitative conversion to selenite in HCI min

30 30 30

HCI only 180 $9

, I,

potable water 5 dil. KMn04 8 acid digests 30

30 15 10 1 5 4

10 20 30

120 20

Cont.flow S

"C [HCII 20 3M [362] 105 4M [363]

85 5M [363] 65 6M [363]

boiling 4M (1811

95 5M [364] , 6M [210]

90 SM [365] 95 SM [6,366] 80 6M [I871

boiling 4M * [205] boiling 4M [I881 boiling 6M [368] boiling 6M [I991

80 6M 1181

boiling water bath 6M [471 boiling water bath 6M [411 heated coil 7.SM [I981

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Table 1) NUCLEAR REACTIONS OF SELENIUM ISOTOPES [54,309,310]

Isotope Natural thermal neutron

74-Se 0.87 YO 30 barns 76-Se 9.02 YO 21 78-Se 23.52 YO 0.33

0.05 80-Se 49.82 % 0.5

0.08 82-Se 9.19 YO 0.004

0.04

abundance cross section product isotope 75-Se 77m-Se 79m-Se 79-Se 8 1 -Se 8 1 m-Se 83-Se 83m-Se

half- principal life y-lines (keV) 120.4 d 12 111 36/265/279/401 17.5 s 161 3.91 m 96 weak

65000 a no y 18.6 m 280 weak 56.8 m 103 weak 25 m 360/520/830/13 10 70 s 650/10 10/2020

Some reactions with epithermal neutrons: 77-Se 7.65 % (n,p) 77-As 39 h 78-Se 23.52 YO (n,2n) 77m-Se 17.5 s 80-Se 49.82 YO 0.04 (n,cr) 77-Ge 11.3 h 82-Se 9.19 % 1.5 (n,2n) 81m-Se 56.8 m

Table 2) NUCLIDES INTERFERING WITH THE y-RADIATION EMISSION SPECTRUM OF 75-SE

75-Se line interferent keV line keV

121.1 Eu 121 8 Ba 1277

135 9 Hf 133 I

Yb 1307 Mo 1404

264 5 Ta 264 1 Cd 2609

275 5 Hg 2792

400 7 Au 411

resulting nuclide 152-Eu 131-Ba 181-Hf

169-Yb

182-Ta

203-Hg

99-TC

115-Cd

l 9 8 - A ~

half-I ife

12.7 y 1 1 5 d 42 5 d

3 1 8 d 66 7 h 115 d 53 5 h 46 6 d

2 7 d

g Se/g interferent Ref thermal n epith n

97 22 [3111 0 2 0 2 [3111 7 2 3 [3111

[3131

[3 I4,3 151 1 5 3 7 [311,313] 0 4 0 6 [3111

5 I 131 11

[3 121

2 4 1 7 [311]

[3161 [91