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Hyphenated techniques applied to environmental speciation studies
trends in analyticalchemistry, vol. 11, no. 1, 1992
,ling and Detection in High-performance Liquid Chromatography (J. Chromatogr. Library Vol. 39A), Elsevier, Amsterdam, 1988, pp. 5-80. 2 E.A. Hogendoorn, C.E. Goewie and P. van Zoonen, Fresenius. J. Anal. Chem., 339 (1991) 348. 3 E.A. Hogendoorn, A.P.J.M. de Jong, P. van Zoonen and U.A.Th. Brinkman, J. Chromatogr., 511 (1990) 243. 4 E.A. Hogendoorn, P. van Zoonen and U.A.Th. Brinkman, Chromatographia, 31 (1991) 285. 5 E.A. Hogendoorn and P. van Zoonen, Med. Fac. Landbouww. Ri]ksuniv. Gent, (1991) in press. 6 E.A. Hogendoorn and P. van Zoonen, Med. Fac. Landbouww. Ri]ksuniv. Gent, 55(3b) (1990) 1275. 7 E.A. Hogendoorn, R. Hoogerbrugge, C.E. Goewie, P. van Zoonen and P.J. Schoenmakers,J. Chromatogr., 552 (1991) 113. 8 K. Grob, On-line coupled LC-GC, Hiithig Verlag, Heidelberg, 1991. 9 H.-G. Schmarr, A. Mosandl and K. Grob, J. High Resolut. Chromatogr., 12 (1989) 721. 10 R. Majors, J. Chromatogr. Sci., 18 (1980) 571. 11 K.A. Ramsteiner, J. Chromatogr., 393 (1987) 123.
17
12 I.A. Mostert and K.A. Ramsteiner, J. Chromatogr., 477 (1989) 359. 13 V. H~ikkinen, K. Grob and Ch. Biirgi, J. Chromatogr., 473 (1989) 353. 14 F.A. Marls, E. Noroozian, R.R. Otten, R.C.J.M. van Dijck, G.J. de Jong and U.A.Th. Brinkman, J. High Resolut. Chromatogr., 11 (1988) 197. 15 K. Grob, E. Miiller and W. Meier, J. High Resolut. Chromatogr., 10 (1987) 416. 16 E.A. Hogendoorn, G.R. van der Hoff and P. van Zoonen, J. High Resolut. Chromatogr., 12 (1989) 784. 17 G.R. van der Hoff, S.M. Gort, R.A. Baumann, U.A.Th. Brinkman and P. van Zoonen, J. High Resolut. Chromatogr., 14 (1991) 465. 18 P. van Zoonen, G.R. van der Hoff and E.A. Hogendoorn, J. High Resolut. Chromatogr., 13 (1990) 483. Drs. P. van Zoonen, E.A. Hogendoom, G.R. van der Hoff and R.A. Baumann are at the Institute of Public Health and Environmental Protection (RIVM), P.O. Box 1, 3720 BA Bilthoven, Nether/ands.
Hyphenated techniques applied to environmental speciation studies O. F. X. Donard and F. M. Martin
Talence, France Improved understanding of the environmental behsviour, fate and Impact of trace metals and, more spaciflcelly, organometallic compounds requires techniques that are both highly selective and sensitive. Hybridization of separation techniques such as gas or ,quid chromatography coupled to a wide variety of atomic spectrometry detectors has allowed significant progress to be made in the last decade. Evolution of the concept of speciation The concept of speciation was first introduced in 1954 to improve the understanding of the biogeochemical cycling of trace elements in seawater a. It was then difficult to detect individual species. Kinetic and thermodynamic information together with analytical data made it possible to differentiate between oxidized versus reduced, complexed or chelated versus free metal ions in solution and dissolved between particulate species. In addition, the Minamata accident in Japan, induced by the bioaccumulation of mercury as methylmercury (CH3)Hg ÷ in the foodchain, triggered interna0165-9936/92/$05.00.
tional interest in the determination of the methylated forms of mercury rather than the total burden of mercury. Some of the first analytical techniques used were electrochemical methods which differentiated ionic from complexed metal species in solution. Further refinements lead to 'speciation schemes', in which metal species were classified according to their size, charge, polarity, reactivity to specific chemical reagents, or time of chromatographic elution 2. Sequential extractions were carried out in an attempt to define the geochemical association of trace metals with the particulate phase after their solubilization from the matrix by successive chemical reagents 3. Quantitation was either by electrochemical methods or, more commonly, atomic absorption spectrometry. These techniques were complex, slow and lacked sensitivity. The speciation problem has also been addressed using nuclear magnetic resonance spectrometry (NMR). Chemical forms of aluminium were characterized in situ by multinuclear N M R 4. This method is non-destructive but is here also very insensitive. It is beyond the scope of this article to cover the ever increasing number of methods developed to © ElsevierSciencePublishersB.V. Allrightsreserved
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trends in analytical chemistry, vol. 11, no. 1, 1992
address metal speciation. Some of the diverse aspects of speciation analysis have recently been reviewed 5. According to the IUPAC definition, speciation is the process yielding evidence of the atomic or molecular form of an analyte. Application of this concept to environmental issues implies sensitivity. The focus here is on the development of hyphenated techniques that have allowed significant progress to be made in understanding the fate of redox species and organometallic compounds in the environment. The need for direct molecular identification of the organo-metal entity in environmental sample matrices has grown considerably during the last decade. The first direct detection of alkylated forms of lead in gasoline by connecting a gas chromatograph to the nebulizer of an atomic absorption spectrophotometer was reported in 19666. This new approach to speciation, strongly dependent on the interface between separation methods and detectors, had a slow growth at first7, but can now be considered as one of the major trends 8'9.
Speclation as a necessary approach Fig. 1 illustrates the environmental implications resulting from speciation analyses for tin compared with total tin determination by classical atomic absorption spectrometry. Levels of tin in O.D.
b
a
Sample s i z e
100~1
20 ml
£ Io
¢tl
the same marina water sample were determined by two methods. Fig. la presents the recording of tin atomization during the classical determination of the total tin content by graphite furnace atomic absorption spectroscopy (GFAAS). Fig. lb is the chromatogram of the same sample after speciation with a coupled technique using hydride generation, cryogenic trapping, chromatographic separation and determination by atomic absorption in an electrothermally heated quartz cell (H-CT-GC-QFAAS). The total tin content in the sample is in the ng/ml range and was found to be slightly higher with the GFAAS instrument than after speciation. In the first case, no assumption can be made about the forms of tin likely to occur in the sample since they are all dissociated during the atomization process. Despite the high levels of tin detected, the GFAAS results give no significant cause for concern as inorganic tin is usually non-toxic. The same sample analyzed with the H - C T - G C QFAAS shows that the forms of tin present were inorganic tin and butyltin species. The lower total tin concentration measured with the H - C T - G C QFAAS system illustrates the difficulty in obtaining full recovery of the species present in the sample. A further limitation is that all species need to be full derivatized (here formation of hydrides) to be detected. Despite these analytical considerations, the presence of a significant peak of tributyltin (TBT, n(faH9)3Sn +) corresponding to less than 10% of the total tin content suggests the occurrence of an environmental problem associated with the area of sampling. TBT is now recognized as being extremely toxic to marine biota. Authorities concerned with environmental regulations are presently debating an acceptable TBT level for the marine environment. Levels of TBT (as Sn) considered to be acceptable to marine biota range between 20 down to 2 ng/11°. These levels are far beyond the usual capabilities of classical instrumentation both in terms of selectivity as well as sensitivity.
Hyphenated techniques
L
Time (min) 0 ~ ' ~ . 5
Fig. 1. Determination of the tin content in a marine water sample. (a) Total tin determination by GFAAS. (b) Speciation of tin compoundsby H - C T - G C - Q F A A S . (O. F. X. Donard, unpublished data.)
Speciation techniques address only a fraction of the total metal present in the sample and therefore require ultra-sensitive methods (ng/1 range in water and ng/g in solid matrices). This problem has been solved most frequently by coupling chromatographic separation techniques to sensitive and selective detectors (Figs. 2 and 3). The determination of chemical species at ultra-trace levels in environmental samples has renewed the need for a
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trends in analytical chemistry, vol. 11, no. 1, 1992
5PECIATION
TECHNIQUE.5
5eparahon/Concentration
Detection
I F I A , HPLC
I
FAA5
H-C T-GC
QFAA5
]
GC, CGC ICP-AE5
ICP-M5
I
Fig. 2. The main hyphenated techniques interfaced with atomic spectrometrydetection.
strong analytical chain prior to instrumental analysis. Contamination-free sampling is of the outmost importance since levels to be detected are extremely low. The stability of the chemical species during sample storage is now probably the most critical step for environmental speciation analysis11. Redox species are particularly unstable. Methylation, degradation or loss via volatilization of some organometallic species may adversely affect the determination of the organometallic compounds initially present in the sample. Finally, sample pretreatment can significantly modify the yield of species detected in the sample. In most cases, analytical speciation schemes rely on the combination of three basic stages and their interfacing design at the instrumental level: • analyte preconcentration • separation (chromatographic or differential concentration mode) • selective detection (single or multi-element) Table I summarizes all hyphenated techniques 5peciation techniques coupled to atomic spectroscopy detectors 10
sample volume lpg I - lOml 10 - 100 ml I
-
100 MI
102
1rig
I
H- CT/GC - QFAA$
I
-
102e~ments,spec~s ]
Pb, Cu, Cd, C~Fe, Hg,Sb,As Sn, Pb, As, I'~, 5b. Se
I
~:-OFAAs
10 _ 100 ~al 10
10
FIA-AAS
I
Pb, Hg, 5e
I HPLC-GFAA5 Sn, Pb, As, Hg
100 ~tl
H PLC - FAAp-~'E~ Pb,
Det eclion
Sn
limits
Fig. 3. Sensitivityranges of the main hyphenatedtechniques interfacedwith atomicspectrometrydetection.
used and their applications to the speciation of Se, Sn, Pb, As, Sb, and Hg. The major developments in hyphenation for speciation analysis are at present primarily focused on the determination of redox species of metals and/or on the discrimination between inorganic and organometallic species present in environmental samples. The different options developed for speciation are the hyphenated techniques which use on-line sample pretreatment and concentration as opposed to analytical methods where sample pretreatment and concentration are performed off-line from the instrumental set-up. The former includes flow-injection analysis (FIA) and hydride generation (H) methods coupled to cryogenic trapping (CT), the latter covers all classical gas (GC) or liquid chromatography (LC) techniques coupled to a wide variety of detectors 12'13. Hyphenated techniques using on-line sample preconcentration
F/owinjectionanalysis FIA has never been primarily applied to speciation analysis. Speciation analysis with this technique should rather be thought of as a differential determination method using on-line discriminating chemical reactions rather than being based oll true chromatographic separation properties. The sensitivity required for speciation analysis is obtained by direct selective preconcentration on microcolumns, low contamination, and the possibility of on-line matrix pretreatment leading to an overall excellent reproducibility (0.5-1%). Analytes are concentrated from milliliters of the sample to the few microliters which are injected into the detector, thereby allowing concentration factors of 50 to 100. This preconcentration ability can be illustrated with the combination of on-line sorbent extraction/preconcentration procedures applied to the difficult discrete GFAAS detection 14. Speciation conditions can be achieved by several continuous separation techniques such as dialysis, gas diffusion, ion exchange, liquid-liquid extraction or reaction with redox reagents_15 . The high affinity of amphoteric activated alumina can be used to separate oxyanions or cations undel acidic or basic conditions respectively (Fig. 4) 16. Detectors adapted to flow injection techniques in trace metal speciation have to match the continuous flow of the technique. Flame atomic absorption spectrometry (FAAS) and an inductively coupled plasma connected to atomic emission speo
20
trend~" in analytical chemistry, vol. 11, no. l, 1992
TABLE 1. Main hyphenated techniques using chromatography interfaced with atomic spectrometry and applications (adapted from ref. 8) Species
Element
Hyphenated technique Air
Water Sediment
Biol. tissues
Se(IV)/Se(VI), MethylMethylMethylInorg. Se Se(IV)/Se(VI) Se(IV)/Se(Vl)
Se
H-CT-GC-QFAAS GC-QFAAS GC-GFAAS GC-MIP-AES LC-GFAAS HPLC-ICP-AES
+ +
+ +
+
MethylHg(ll) MethylMethyl-
Hg
GC-CVAAS GC-GFAAS GC-MIP-AES HPLC-CVAAS
+
As(III)/As(V), MethylAs(III)/As(V), As(III)/As(V), As(III)/As(V),
Methyl-
As
MethylMethyl-, PhenylMethyl, ASB,ASC
H-CT-GC-QFAAS GC-QFAAS GC-FPD HPLC-GFAAS
I
e0
I'", Cr(lll) ii
I ~
11
I
"
'1
O
e
._c
8O
, I
C
.9
•g
i_ 4o
I
,
J
"J
g "~-g
'' it I
•
.~
Ill I I
[ 11
%111 I
.,Jl,i I i I
Iih.Ii
I
II
I
0
I
I
20
40
Itlllllllll
I
60 Time (s)
+
+
III Cr (VI)
=
+
+
GC-FAAS GC-GFAAS GC-QFAAS HPLC-FAAS HPLC-GFAAS HPLC-MIP-AES HPLC-ICP-AES
•
+ +
+
Pb
v
+
+
Methyl-, Methylethyl-, Ethyl-, Methyl-, Methylethyl-, EthylMethyl-, Methylethyl-, EthylMethyl-, EthylMethyl-, Methylethyl-, EthylMethyl-, Methylethyl-, EthylMethyl-, Methylethyl-, Ethyl
~12 0
+
+
+
H-CT-GC-QFAAS
~160
Blood, flour, leaves + +
+
Sb
200
Standards
H-CT-GC-QFAAS GC-MIP-AES HPLC-H-QFAAS HPLC-GFAAS HPLC-ICP-AES
Sb(lll)/Sb(V), Methyl-
Sn Inorg. Sn, Methyl-, ButylInorg. Sn, Methyl-, Butyl-, PhenylInorg. Sn, Methyl-, Butyl-, PhenylButyl-, Phenyl-
+
8'0
I o Jl I
100
Fig. 4. Speciation of chromium Cr(III)/(VI) by FIA-ICP-AES (emission 267.72 nm). Separation after selective preconcentration and elution from activated alumina microcolumn.
Cr(III)--~Cr(VI) solution, 0.1 mg/I. (From ref. 16.)
Comments
+ +
+
+
+
+
+ + +
+
+ + +
Urine, Sweat
Standards Soils Oil shale, process water
Aerosols Gasoline Gasoline Street dust, aerosols Gasoline Standards Industrial effluent Gasoline, standards
+ + +
Organotin dust
trometry (ICP-AES) have been extensively used. Flow injection methods have been reported for the differentiation of redox species such as Cr(III)/Cr(VI), Fe(II)/Fe(III), As(III)/As(V), Se(IV)/Se(VI). Speciation between inorganic and methyl mercury can also be obtained by the differential affinity of sulphydryl cotton to methyl mercury (Fig. 5) 16. The application of these methods has been mainly directed to the determination of redox species in waters, and sometime in sediments and soil extracts. They are however always limited by potential saturation of the active sites by major ions contained in the matrix. A judicious selection of column packing is therefore needed. Flow injection as a speciation tool is still in its infancy. Among the important future contributions is the possibility of coupling flow injection to highperformance liquid chromatography (HPLC) for on-line sample clean-up, precolumn derivatiza-
21
trends in analytical chemistry, vol. 11, no. 1, 1992
o
E
U "1"
"~
Inorgaric Hg
~
MeHg
t
~
J ~ Time
During the first step, analytes are extracted from the sample matrix by hydride generation under acidic or basic conditions. Hydride generation is now well established for elements such as As, Bi, Sb, Se, Sn, Ge and Te, and it is also efficient for most light alkylated species. In general, the comparison between the pH and pK a of the species shows that the reduction is generally performed at a pH that is a few units below the pK a of the species of interest 2°. The general reaction can be described for alkylated tin compounds21:
i
RxSn(4-x) + NaBH4, H + H+ ~ RxSnH(4-x) + H2 Fig. 5. Reproducibility of FIA-AFS for the speciation of inorganic mercury and methyl mercury with selective preconcentration and elution from a sulphydryl cotton microcolumn. Hg(ll)/MeHg ÷ solution 2 ng/I. (Courtesy C. W. McLeod & W. Jian, Sheffield Polytechnic, UK, presented at the 1990 Winter Conference on Plasma Spectrochemistry, St. Petersburg, USA, January, 1990.)
tion, and the elimination of matrix effects 17. Possibilities of interfacing flow injection to H - C T - G C - Q F A A S also exist and need to be explored. In general, automation of flow injection hyphenated systems will allow higher sample thoughput. Also, if species preconcentration of the sample in the field and stable storage on the same solid support could be achieved under good conditions and later directly eluted in a flow injection hyphenated system, this would change the general prospect of environmental speciation analysis by reducing sample size and analytical errors associated with sample contamination through handling. Preliminary results have been obtained for Cr preconcentration and later speciation in river waters 18.
Hydride generation, cryogenic trapping, chromatographic separation and atomic spectrometry detection This hyphenated technique was first used in 1975 for the determination of methylated forms of selenium in freshwater environments 19. More recently, due to the simplicity and high sensitivity of the method, it has been extensively applied to the determination of butyltin compounds in the environment. It combines the following basic steps: on-line aqueous sample derivatization, preconcentration by cryofocussing, chromatographic separation, detection by AAS in an electrothermally heated quartz furnace and signal processing.
(1)
where x = 1,2,3; and R is methyl-, ethyl- or butyl-. This technique can also differentiate the redox forms of As, Se and Sb as appropriate pH reaction ranges can be selected. Important species of Hg and Pb cannot be directly analyzed by hydride reduction processes. However, the field of application of this technique has now been extended to Hg and Pb as well as to their alkylated derivatives by the use of a new boron alkylating reagent in water (NaBEt4) 22. Hg and Pb were quantitatively ethylated at pH 5 in water according to the following reactions:
Pb2++2
, EtzPb n + . . .
(2)
2 Et2Pb n
~Et4Pb Iv + Pb °
(3)
Me2Pb 2+ + NaBEt 4
~Me2Et2Pb + . . .
(4)
Hg 2 + 2 NaBEt 4
, Et2Hg + . . .
(5)
NaBEt 4
The application of this new derivatization is currently under development. It has been shown to be less sensitive to interferences than hydride generation for the determination of butyltin compounds in complex sediment matrices. The on-line preconcentration and separation steps of derivatized species is one of the major advantages of the H - C T - G C - Q F A A S method. Cryofocussing allows preconcentration factors of 50- to 100-fold and is performed on a small packed chromatographic column coated with a non-polar methylsilicon phase immersed in liquid nitrogen. Separation is achieved upon warming of the trap and species are eluted on the basis of their boiling points as well as their chromatographic properties. This provides a limited separation potential of approximately 3000 plates. The combination of this technique with the highly specific detection cap-
22
trends in analytical chemistrv, vol. II, no. 1. 1992
abilities of AAS using deuterium background correction allows selective recording of inorganic and organometallic forms of the metal present in the sample on the same chromatogram. Fig. 6 presents an extreme effect obtained with sample clean-up of a heavily polluted sediment leachate. Fig. 6a shows the standard chromatogram of the sediment after direct leaching with acetic acid. Fig. 6b displays the increased recovery obtained after clean-up of the leachate. Butyltin compounds are significantly enhanced. Clean-up of the sample also provided evidence of the presence of methylated tin in the sample. The interfacing of the different hyphenation stages is simple, but the system must be able to withstand the high pressure obtained during the hydride generation reaction. Despite its apparent simplicity, this set-up needs to be fine-tuned to obtain high sensitivity. In general, reproducibility with such systems ranges between 5 and 15% (n = 5). Careful automation of each analytical stage should considerably reduce the relative standard deviation routinely obtained. Minimal dead volume is essential for high sensitivity and low tailing of the chromatographic peaks. The continuous gaseous effluent from the chromatographic column is readily atomized in a quartz cell aligned in the beam of an atomic spectrophotometer. Both flame and electrically heated cells are adequate. The addition of H 2 and 0 2 gas in the quartz furnace is critical in obtaining enhanced sensitivity and atomisation mechanisms are based on OH ° free radical processes. This step needs to be developed further in order to achieve maximum sensitivity
m ÷
a
&
=
b ÷
==
£ +
7~ £ ,.t:l ,<
lime ( m i n )
Time (rain)
Fig. 6. (a) Organotin speciation in sediments by H-CT-GCQFAAS after acetic acid (96%) leach. (b) The effect of sample clean-up by a Sep Pak C18microcolumn after conditioning by methanol and acetic acid (96%). (O. F. X. Donard and F. Martin, unpublished data.)
and to control interferences associated with atomisation processes. This inexpensive atomizer is very popular, has seen many designs, and has been applied to several organometallic species of Sn, Se, As, Sb, Pb, and Hg. Inorganic and methylated germanium species can not be dissociated at the low temperature of the QFAAS (1000°C) and are detected by continuous firing of the graphite furnace. Other detectors, such as flame photometric detectors (FPD), provide excellent sensitivity with this set-up but are not selective enough. Atomic fluorescence spectroscopy (AFS) is the preferred detector for mercury analysis. It is more sensitive than AAS and detection limits are at the picogram level for the speciation of inorganic and methylated forms of mercury in the environment 23.
Hyphenated techniques using off-line sample preconcentration Separation by gas chromatography GC has been interfaced successfully with a wide range of detectors. Absolute detection limits obtained with these systems are similar to those using on-line preconcentration since they ultimately use an atomic absorption detector. However, the important difference lies in the initial sample volume. With on-line hyphenated systems, the sample volume can be as large as several hundred milliters if required. With off-line hyphenated systems, only microliter volumes can be injected in the gas chromatograph and therefore preconcentration of the analytes in the aliquot injected is required. Extraction from the sample and preconcentration then needs to be performed off-line in a solvent medium with or without chelating agents. Difficulties exist in the quantitative liquid-liquid extraction of all ionic inorganic and organometallic species. After extraction from the sample, there are several procedures to derivatize and concentrate the analytes prior to chromatographic separation. These procedures include numerous steps. Organometallic compounds can be extracted from the matrix and derivatized by hydride generation. Cryofocussing on glass beads after hydride generation and subsequent GC separation gives the necessary preconcentration required to obtain good sensitivity. In most cases, organolead or organotin compounds react with a Grignard reagent to form volatile tetraalkyl substitutes suitable for chromatography.
23
trends in analytical chemistry, voL 11, no. 1, 1992
Rx(Sn,Pb)(4_x)+ R'MgX Rx(Sn,Pb)R'(4-x)
(6)
solvent
where x = 1, 2, 3; and R is inorg. Sn, methyl-, ethyl-, butyl-, or phenyl. For lead R' can be a propyl-, butyl-, or phenylgroup. For tin, a larger variety of R' substituents have been used such as methyl-, butyl-, pentyl-, and hexyl-groups. After derivatization, the excess Grignard reagent is removed by addition of sulphuric acid and use of a drying agent such as anhydrous CaCl 2. Finally, the samples are concentrated under a gentle stream of nitrogen. These numerous procedures preconcentrate organometallic compounds from large samples thus bringing them into the dynamic range required for speciation studies. The detection limits are in the ng/1 or ng/g range and are suitable for most environmental samples. An important drawback of the technique is the long and tedious off-line extraction/derivatization/preconcentration procedures which, in addition, may be a source of error due to the loss of analytes or of the internal standard. Simultaneous derivatization/liquid-liquid extraction has been applied successfully to minimize these numerous sample pretreatment steps. Recently, on-column derivatization has also been developed. Pellets of NaBH 4 or NaBEt 4 are added on top of the packed column in the injection port. Direct injection of the analytes onto the top of the column results in quantitative hydritization or ethylation of the organometallic species 24'25. Organometallic compounds are then separated on classical chromatographic systems. Interfacing is a critical step in achieving good separation. Transfer lines from the GC to the detectors should be as short as possible, and they should be heated to avoid analyte condensation and decomposition. High-resolution capillary gas chromatography (HRCGC) has probably indirectly increased the sensitivity of the set-up. Increased separation of compounds likely to coelute with organometallic species prevents their competition later in the atomisation and detection processes. The sharp peaks obtained make a heavy demand on the detector's speed-of-data acquisition capacities. Several types of detectors have been coupled to GC for trace speciation. In most cases, the solvent is a potential problem for the detectors used. The most popular detector to date is certainly the electrothermally heated quartz atomizer. Although not highly specific, FPD detectors fitted with a 610 filter to monitor the molecular emission of the
S n - H in nitrogen/hydrogen diffusion flames are gaining increasing popularity, particularly when coupled to capillary GC for the detection of organotin compounds. AES has also been widely used on simple solution matrix samples. AES has a larger dynamic range than AAS. The high cost of the multi-elemental plasma emission detector has limited its use as a chromatographic detector. The increased use of the helium TM010 microwave cavity, developed by Beenakker to operate at atmospheric pressure, has allowed simple interfacing with GC [GC-microwave induced plasma(MIP)-AES]. This plasma source requires low energy and generates high electron temperatures, giving high spectral intensity. It is used for AES of a wide number of elements such as N, P, S, C, Br, CI, H, Si, Hg, Sn and Pb. Detection limits are in the picogram range. It has been basically used for the speciation of alkylated lead and mercury compounds in the environment. Atomic fluorescence is another mode of detection worth mentioning although seldom used. It can provide multi-elemental determination 26. Mass spectrometry (MS) connected to GC is often used for confirmation of the occurrence of new organometallic species found in the environment. The sensitivity of small bench-top systems is too low (ng level) to be used routinely in the full scan mode. However, when used in the selected ion monitoring mode (SIM), detection limits can be improved by 100- to 1000-fold. The continuous improvement in sensitivity of quadrupole instrumentation will certainly allow it to be increasingly used in environmental speciation analysis.
Separationby liquid chromatography Liquid chromatography (LC), at present mainly HPLC, is another popular method for the separation of metallic species prior to detection. This trend does not compete with the other hyphenated techniques mentioned above but rather expands the type of chemical and physical species concerned in speciation analysis. It offers several advantages compared to GC techniques. HPLC methods allow the separation of species without derivatization. Furthermore, the wide variety of chromatographies (adsorption, ion-exchange, gel permeation, normal and reversed-phase chromatography) allows the separation of ions, volatile species, high molecular weight organometallics as well as complex, large-size biological species. Important efforts have been focused on the speciation of arsenic compounds in soil extracts and
24
biological tissues. The polarity of inorganic and most organo-arsenic species makes them amenable to both ion-exchange and reversed-phase HPLC separation. The forms of arsenic concerned are arsenite, arsenate, monomethylarsonate (MMA), dimethylarsinate (DMA), arsenobetaine (ASB), arsenocholine (ASC) and arseno-sugars. The last three species are not volatilized by hydride generation. Organotin and organomercury compounds can also be separated with HPLC methods. The possibility of volatilization by online hydride generation after HPLC separation has already been explored for mercury and tin 27. HPLC coupled techniques are mainly used for the speciation of organometallic or metal-organic molecules in soils, oils or biological tissues after the preliminary clean-up and concentration steps. LC as a separatory tool has also been applied to a wider range of metal-organic compounds such as metal-amino acid complexes or protein-bound metals in biological fluids28'29. Separation of metal chelates and simultaneous detection by AES is also possible 3°. Hyphenation between LC methods and atomic spectrometry detection mostly results in low sensitivity instrumentation. Cryogenic trapping methods are 1000-fold more sensitive than HPLC based techniques. The interface is one of the conditional factors for sensitivity. Due to low efficiency in the nebulization and desolvation of the mobile phase, the major problem is the conversion of analytes to volatile forms which are easily detectable. Another problem lies in matching the eluent flow with the aspiration rate of the nebulizer. Finally, the organic solvent is usually a source of interference in the detector. FAAS was the first candidate for coupling. The low sensitivity of this technique has led several researchers to use GFAAS because of the latter's excellent detection limits. The difficulty in interfacing here lies in the compatibility between the continuous elution of the mobile phase and discontinuous nature of graphite atomisation detection. HPLC-GFAAS systems were developed for the speciation of organometallic compounds of arsenic and tin (Fig. 7) 31'32. Eluents from reversedphase columns may induce a high background molecular absorption requiring the use of the Zeeman effect correction rather than the deuteriumlamp correction. Speciation by LC coupled to plasma systems is 20 times more sensitive than with FAAS and was first demonstrated more than a decade agO33. I C P AES has proven to be relatively interference-free
trends in analytical chernistry, vol. 11, no. 1, 1992
'A.u.) 375
b
250
d
0
aLhl ,ll,
....
IIII kJ
lo 2'o 3o
h,
Time (min)
Fig. 7. Speciation of butyltin compounds by on-line HPLCGFAAS. a = tetrabutyltin (100 mg/I), b = dibutyltin dichloride (400 mg/I), c = tributyltin chloride (400 mg/I), d = monobutyltin trichloride (400 mg/I). Separation on a 250 × 4 mm I.D. Nucleosil 5 CN column. (Courtesy M. Astruc, A. Astruc, R. Pinel, Universit6 de Pau, France.)
for the monitoring of arsenic species and is widely used. Despite their lower sensitivity in comparison to ICP-AES detectors, direct current plasma jets (DCP) are less affected by the solvent and are gaining increasing popularity as LC detectors. A further development has been the coupling of HPLC systems to ICP-MS. Some of the first interfaces realized allowed the determination of meth-
4
5
I
Time4 {rain }
8
Fig. 8. Speciation of arsenic compounds by H P L C - I C P - M S ) . 1 = arsenocholine (31 #g/I), 2 = arsenobetaine (37/~g/I), 3 = As(Ill) (76/tg/I), 4 = dimethylarsinic acid (47/~g/I), 5 = monomethylarsonic acid (41/~g/I), 6 = As(V) (65/~g/I). Separation was performed on an Hamilton PRPX 100 column (250 mm x 4 mm I.D.) Injection loop: 20/A. (Courtesy A. Lamotte, C. Demesmay, M. Olle, Service Central d'Analyse CNRS, Lyon, France.)
25
trends in analytical chemistry, vol. I1, no. 1, 1992
ylmercury34 or dimethylarsinic acid and arsenobetaine 35 in fish extracts. By choosing appropriate chromatographic conditions, extensive speciation of arsenic compounds can be obtained (Fig. 8). Progress in this field can certainly be expected from the coupling of the microbore HPLC system with microconcentric nebulisers and the highly sensitive multielement ICP-MS detector. Future trends
If speciation analysis were only restricted to separation and sensitivity, the merging of new techniques would considerably extend its potential. The combination between supercritical extraction and separation (supercritical fluid chromatography, SFC) techniques would extend separation capabilities in areas where neither GC nor HPLC may be suitable. Detectors would greatly benefit from the direct introduction ef the sample in gaseous form. Supercritical fluid transport to the ICP allows sample introduction with almost 100% efficiency. Interface design is one of the most critical areas where progress is needed to improve sensitivity of detectors coupled to liquid separation techniques. The progress made with ICP-MS detectors is an indication of one of the major future trends of development in speciation analysis. It offers a new range of sensitivity that needs to be explored by tandem association with either GC or HPLC. Ultimate detection limits can also be obtained with laser-enhanced ionization in GC or LC effluents. Laser-enhanced ionization as an element specific detector of GC effluents using an excimer laser and a time-of-flight mass detector can reach absolute detection limits in the attogram (10 -18 g) range. Ion-trap mass spectrometry has also been successfully interfaced with separation techniques such as GC, LC and SFC. It then performs like any other mass spectrometer but provides higher sensitivity. When independent from the coupling and under optimum operating conditions, detection limits are also in the attomole range. The mass range for molecular weight determination is large and there are capabilities for mixture analysis and structural elucidation. The historical development of speciation analysis can be summarized by considering the improvement in determining organometallic molecules of increasing size in water samples and large metalorganic biopolymers in biological systems. Capillary electrophoresis is now applied to the speciation of organometallic forms of arsenic (Fig. 9). It can also be coupled to MS detection. The in-
As203
DMA
-t
As20s MMA
/ a,
1
"
Time (min)
Fig. 9. Speciation of arsenic compounds by Free Solution Capillary Electrophoresis. Detection by UV at 190 nm. Sample concentration, 100 mg/I, injection volume 9 nl. (Courtesy M. Leroy, EHICS, Strasbourg, France.)
creasing use of LC-MS will yield new information on the identity of large metal-organic molecular interactions (e.g. identification of porphyrins, large metal and organometallic complexes). Recently, electrospray interfacing for coupling ionexchange chromatography to mass spectrometry (MS) has been reported to yield effective separation and good sensitivity for the determination of arsenobetaine in dog fish muscle 36. These applications will also benefit from the ongoing developments of heavier instrumentation in organic MS ~'. However, this instrumentation is expensive and will not be easily accessible to many laboratories concerned with environmental problems. One of the main reasons for the slow development in metal speciation studies is the lack of available commercial instrumentation. On a simple basis of cost and sample throughput, refining the integration of FIA in existing hybrid techniques represents another more affordable trend. FIA will allow on-line sample preconcentration and pretreatment. It will also facilitate post-column reactions, such as the addition of matrix modifiers prior to detection, on existing hyphenated systems. Atomic absorption will for some time still be the detector of choice owing to its low cost, high specificity, sensitivity with a quartz electrothermal atomizer cell and wide range of interfacing modes. GC coupled to AES with a microwave induced plasma is now reaching its full range of applications and is commercially available.
trendsin analyticalchernistry, vol. 11, no. 1, 1992
26
in general, high levels of automation are required since the essence of speciation analysis is based on the consecutive yield of physical-chemical reactions leading to quantitation of the analytes. However, it should be borne in mind that speciation analysis requires a strong analytical chain prior to determination. Tandem association is certainly a field that can be instrumentally well mastered. Nevertheless, sample collection, sample concentration and pretreatment are unavoidable steps that needs to be carefully controlled to obtain reliable analyses. Much effort still has to be invested in these preliminary steps. Speciation is now mainly devoted to environmental analysis. Major progress can be expected in other areas such as water treatment, the pharmaceutical industry, biotechnology and medicine.
Acknowledgements The authors would like to thank H. Budzinski, P. Garrigues, F. Fenter and C. McLeod for their help with the manuscript. C. McLeod, M. Astruc, A. Lamotte and M. Leroy are gratefully acknowledged for their contribution.
References 1 E. D. Goldberg, J. Geol., 62 (1954) 249. 2 G. E. Batley (Editor), Trace Element Speciation: Analytical Methods and Problems, CRC Press, Boca Raton, FL, 1989. 3 A. Tessier, P. G. C. Campbell and M. Bisson, Anal. Chem., 51 (1979) 844. 4 P. J. Slader, in M. Bernhard, F. E. Brinkman and P. J. Slader (Editors), The Importance of Chemical 'Speciation' in Environmental Processes, Springer, Berlin, 1986, p. 563. 5 W. Lund, Fresenius' J. Anal. Chem., 337 (1990) 557. 6 B. Kolb, G. Kemmner, F. H. Schesler and E. Wiedeking, Fresenius' Z. Anal. Chem., 221 (1966) 166. 7 J. C. Van Loon, Anal. Chem., 51 (1979) 1139A. 8 R. M. Harrison and S. Rapsomanikis (Editors), Environ-
9 10 11 12 13
mental Analysis Using Chromatography Interfaced with Atomic Absorption Spectrometry, Ellis Horwood, Chichester, 1989. Y. K. Chau and P. T. S. Wong, Fresenius' J. Anal. Chem., 339 (1991) 640. M. J. Waldock, J. E. Thain and M. E. Waite, Appl. Organomet. Chem., 1/4 (1987) 287. Ph. Quevauviller and O. F. X. Donard, Fresenius' J. Anal. Chem., 339 (1991) 6. L. Ebdon, S. Hill and R. W. Ward, Analyst (London), 111 (1986) 1113. L. Ebdon, S. Hill and R. W. Ward, Analyst (London), 112 (1987) 1.
14 Z. Fang, M. Sperling and B. Welz, J. Anal. At. Spectrom., 5 (1990) 639. 15 M. Valcarcel and M. D. Luque de Castro, Flow injection Analysis, Principles and Applications, Ellis Horwood, Chichester, 1987. 16 A.G. Cox, A.J. Cook and C.W. McLeod, Analyst (London), 110 (1985) 331. 17 M. D. Luque de Castro, Mikrochim. Acta, submitted for publication. 18 A. G. Cox and C. W. McLeod, Mikrochim. Acta, submitted for publication. 19 Y. K. Chau, P. T. S. Wong and P. D. Goulden, Anal. Chem., 47 (1975) 2279. 20 M. O. Andreae, in C. S. Wong, E. Boyle, K. W. Bruland, J. D. Burton and E. D. Goldberg (Editors), Trace Metals in Sea Waters, Plenum Press, New York, 1983. 21 O. F. X. Donard, S. Rapsomanikis and J. H. Weber, Anal. Chem., 58 (1986) 772. 22 S. Rapsomanikis, O. F. X. Donard and J. H. Weber, Anal. Chem., 58 (1986) 35. 23 N. Bloom, Can. J. Fish. Aquat. Sci., 46 (1989) 1131. 24 S. Clark, J. Ashby and P. J. Craig, Analyst (London), 112 (1987) 1781. 25 J. Ashby, S. Clark and P. J. Craig, J. Anal. At. Spectrom., 3 (1988) 735. 26 A. D'Ulivo and P. Papoff, J. Anal At. Spectrom., 1 (1986) 479. 27 D. T. Burns, F. Glockling and M. Harriot, Analyst (London), 106 (1981) 921. 28 N. Khan and J. C. Van Loon, J. Liq. Chromatogr., 2 (1979) 23. 29 P. E. Gardiner, P. Braetter, V. E. Neggretti and G. Schulze, Spectrochim. Acta B, 38 (1983) 427. 30 J. C. Van Loon, J. Lichjaw and B. Radziuk, J. Chromatogr., 136 (1977) 301. 31 F. E. Brinckman, K. L. Jewett, W. P. Iverson, K. J. Irgolic, K. C. Ehrhardt and R. A. Stockton, J. Chromatogr., 191 (1980) 31. 32 R. Pinel, M. Z. Benabdallah, A. Astruc, M. Pautin-Gautier and M. Astruc, Analusis, 12 (1984) 344. 33 M. Morita and T. Uehiro and K. Fuwa, Anal. Chem., 52 (1980) 351. 34 D. S. Bushee, Analyst (London), 113 (1988) 1167. 35 D. Beauchemin, K. W. M. Siu, J. W. McLaren and S. S. Berman, J. Anal. At. Spectrom., 4 (1989) 285. 36 K. W. Sui, R. Guevremont, J. C. Y. Leblanc, G. J. Gardner, and S. S. Berman, J. Chromatogr., 554 (1991) 27. 37 M. Linscheid, Fresenius' J. Anal. Chem., 337 (1990) 648.
i,( ~ X. Donard is a CNRS research scientist at th~Laboratoire de~Pt~6~f~hysique et Photochimie Molecu/air~ at the University of Bordeaux (France)J His interests are in analytical chemistry applied to chemida/ oceanography. He develops analytical methods by interfacing gas chromatography techniques to atomic spectrometry. Fabienoe M. Martin is a PhD student in Chemical Physics applied to the Environment in the same laboratory. She is doing a PhD in analytical chemistry on the hyphenation between gas chromatography and atomic spectrometry detectors such as atomic absorption or inductively coupled plasma-mass spectrometry.
,ling and Detection in High-performance Liquid Chromatography (J. Chromatogr. Library Vol. 39A), Elsevier, Amsterdam, 1988, pp. 5-80. 2 E.A. Hogendoorn, C.E. Goewie and P. van Zoonen, Fresenius. J. Anal. Chem., 339 (1991) 348. 3 E.A. Hogendoorn, A.P.J.M. de Jong, P. van Zoonen and U.A.Th. Brinkman, J. Chromatogr., 511 (1990) 243. 4 E.A. Hogendoorn, P. van Zoonen and U.A.Th. Brinkman, Chromatographia, 31 (1991) 285. 5 E.A. Hogendoorn and P. van Zoonen, Med. Fac. Landbouww. Ri]ksuniv. Gent, (1991) in press. 6 E.A. Hogendoorn and P. van Zoonen, Med. Fac. Landbouww. Ri]ksuniv. Gent, 55(3b) (1990) 1275. 7 E.A. Hogendoorn, R. Hoogerbrugge, C.E. Goewie, P. van Zoonen and P.J. Schoenmakers,J. Chromatogr., 552 (1991) 113. 8 K. Grob, On-line coupled LC-GC, Hiithig Verlag, Heidelberg, 1991. 9 H.-G. Schmarr, A. Mosandl and K. Grob, J. High Resolut. Chromatogr., 12 (1989) 721. 10 R. Majors, J. Chromatogr. Sci., 18 (1980) 571. 11 K.A. Ramsteiner, J. Chromatogr., 393 (1987) 123.
17
12 I.A. Mostert and K.A. Ramsteiner, J. Chromatogr., 477 (1989) 359. 13 V. H~ikkinen, K. Grob and Ch. Biirgi, J. Chromatogr., 473 (1989) 353. 14 F.A. Marls, E. Noroozian, R.R. Otten, R.C.J.M. van Dijck, G.J. de Jong and U.A.Th. Brinkman, J. High Resolut. Chromatogr., 11 (1988) 197. 15 K. Grob, E. Miiller and W. Meier, J. High Resolut. Chromatogr., 10 (1987) 416. 16 E.A. Hogendoorn, G.R. van der Hoff and P. van Zoonen, J. High Resolut. Chromatogr., 12 (1989) 784. 17 G.R. van der Hoff, S.M. Gort, R.A. Baumann, U.A.Th. Brinkman and P. van Zoonen, J. High Resolut. Chromatogr., 14 (1991) 465. 18 P. van Zoonen, G.R. van der Hoff and E.A. Hogendoorn, J. High Resolut. Chromatogr., 13 (1990) 483. Drs. P. van Zoonen, E.A. Hogendoom, G.R. van der Hoff and R.A. Baumann are at the Institute of Public Health and Environmental Protection (RIVM), P.O. Box 1, 3720 BA Bilthoven, Nether/ands.
Hyphenated techniques applied to environmental speciation studies O. F. X. Donard and F. M. Martin
Talence, France Improved understanding of the environmental behsviour, fate and Impact of trace metals and, more spaciflcelly, organometallic compounds requires techniques that are both highly selective and sensitive. Hybridization of separation techniques such as gas or ,quid chromatography coupled to a wide variety of atomic spectrometry detectors has allowed significant progress to be made in the last decade. Evolution of the concept of speciation The concept of speciation was first introduced in 1954 to improve the understanding of the biogeochemical cycling of trace elements in seawater a. It was then difficult to detect individual species. Kinetic and thermodynamic information together with analytical data made it possible to differentiate between oxidized versus reduced, complexed or chelated versus free metal ions in solution and dissolved between particulate species. In addition, the Minamata accident in Japan, induced by the bioaccumulation of mercury as methylmercury (CH3)Hg ÷ in the foodchain, triggered interna0165-9936/92/$05.00.
tional interest in the determination of the methylated forms of mercury rather than the total burden of mercury. Some of the first analytical techniques used were electrochemical methods which differentiated ionic from complexed metal species in solution. Further refinements lead to 'speciation schemes', in which metal species were classified according to their size, charge, polarity, reactivity to specific chemical reagents, or time of chromatographic elution 2. Sequential extractions were carried out in an attempt to define the geochemical association of trace metals with the particulate phase after their solubilization from the matrix by successive chemical reagents 3. Quantitation was either by electrochemical methods or, more commonly, atomic absorption spectrometry. These techniques were complex, slow and lacked sensitivity. The speciation problem has also been addressed using nuclear magnetic resonance spectrometry (NMR). Chemical forms of aluminium were characterized in situ by multinuclear N M R 4. This method is non-destructive but is here also very insensitive. It is beyond the scope of this article to cover the ever increasing number of methods developed to © ElsevierSciencePublishersB.V. Allrightsreserved
18
trends in analytical chemistry, vol. 11, no. 1, 1992
address metal speciation. Some of the diverse aspects of speciation analysis have recently been reviewed 5. According to the IUPAC definition, speciation is the process yielding evidence of the atomic or molecular form of an analyte. Application of this concept to environmental issues implies sensitivity. The focus here is on the development of hyphenated techniques that have allowed significant progress to be made in understanding the fate of redox species and organometallic compounds in the environment. The need for direct molecular identification of the organo-metal entity in environmental sample matrices has grown considerably during the last decade. The first direct detection of alkylated forms of lead in gasoline by connecting a gas chromatograph to the nebulizer of an atomic absorption spectrophotometer was reported in 19666. This new approach to speciation, strongly dependent on the interface between separation methods and detectors, had a slow growth at first7, but can now be considered as one of the major trends 8'9.
Speclation as a necessary approach Fig. 1 illustrates the environmental implications resulting from speciation analyses for tin compared with total tin determination by classical atomic absorption spectrometry. Levels of tin in O.D.
b
a
Sample s i z e
100~1
20 ml
£ Io
¢tl
the same marina water sample were determined by two methods. Fig. la presents the recording of tin atomization during the classical determination of the total tin content by graphite furnace atomic absorption spectroscopy (GFAAS). Fig. lb is the chromatogram of the same sample after speciation with a coupled technique using hydride generation, cryogenic trapping, chromatographic separation and determination by atomic absorption in an electrothermally heated quartz cell (H-CT-GC-QFAAS). The total tin content in the sample is in the ng/ml range and was found to be slightly higher with the GFAAS instrument than after speciation. In the first case, no assumption can be made about the forms of tin likely to occur in the sample since they are all dissociated during the atomization process. Despite the high levels of tin detected, the GFAAS results give no significant cause for concern as inorganic tin is usually non-toxic. The same sample analyzed with the H - C T - G C QFAAS shows that the forms of tin present were inorganic tin and butyltin species. The lower total tin concentration measured with the H - C T - G C QFAAS system illustrates the difficulty in obtaining full recovery of the species present in the sample. A further limitation is that all species need to be full derivatized (here formation of hydrides) to be detected. Despite these analytical considerations, the presence of a significant peak of tributyltin (TBT, n(faH9)3Sn +) corresponding to less than 10% of the total tin content suggests the occurrence of an environmental problem associated with the area of sampling. TBT is now recognized as being extremely toxic to marine biota. Authorities concerned with environmental regulations are presently debating an acceptable TBT level for the marine environment. Levels of TBT (as Sn) considered to be acceptable to marine biota range between 20 down to 2 ng/11°. These levels are far beyond the usual capabilities of classical instrumentation both in terms of selectivity as well as sensitivity.
Hyphenated techniques
L
Time (min) 0 ~ ' ~ . 5
Fig. 1. Determination of the tin content in a marine water sample. (a) Total tin determination by GFAAS. (b) Speciation of tin compoundsby H - C T - G C - Q F A A S . (O. F. X. Donard, unpublished data.)
Speciation techniques address only a fraction of the total metal present in the sample and therefore require ultra-sensitive methods (ng/1 range in water and ng/g in solid matrices). This problem has been solved most frequently by coupling chromatographic separation techniques to sensitive and selective detectors (Figs. 2 and 3). The determination of chemical species at ultra-trace levels in environmental samples has renewed the need for a
19
trends in analytical chemistry, vol. 11, no. 1, 1992
5PECIATION
TECHNIQUE.5
5eparahon/Concentration
Detection
I F I A , HPLC
I
FAA5
H-C T-GC
QFAA5
]
GC, CGC ICP-AE5
ICP-M5
I
Fig. 2. The main hyphenated techniques interfaced with atomic spectrometrydetection.
strong analytical chain prior to instrumental analysis. Contamination-free sampling is of the outmost importance since levels to be detected are extremely low. The stability of the chemical species during sample storage is now probably the most critical step for environmental speciation analysis11. Redox species are particularly unstable. Methylation, degradation or loss via volatilization of some organometallic species may adversely affect the determination of the organometallic compounds initially present in the sample. Finally, sample pretreatment can significantly modify the yield of species detected in the sample. In most cases, analytical speciation schemes rely on the combination of three basic stages and their interfacing design at the instrumental level: • analyte preconcentration • separation (chromatographic or differential concentration mode) • selective detection (single or multi-element) Table I summarizes all hyphenated techniques 5peciation techniques coupled to atomic spectroscopy detectors 10
sample volume lpg I - lOml 10 - 100 ml I
-
100 MI
102
1rig
I
H- CT/GC - QFAA$
I
-
102e~ments,spec~s ]
Pb, Cu, Cd, C~Fe, Hg,Sb,As Sn, Pb, As, I'~, 5b. Se
I
~:-OFAAs
10 _ 100 ~al 10
10
FIA-AAS
I
Pb, Hg, 5e
I HPLC-GFAA5 Sn, Pb, As, Hg
100 ~tl
H PLC - FAAp-~'E~ Pb,
Det eclion
Sn
limits
Fig. 3. Sensitivityranges of the main hyphenatedtechniques interfacedwith atomicspectrometrydetection.
used and their applications to the speciation of Se, Sn, Pb, As, Sb, and Hg. The major developments in hyphenation for speciation analysis are at present primarily focused on the determination of redox species of metals and/or on the discrimination between inorganic and organometallic species present in environmental samples. The different options developed for speciation are the hyphenated techniques which use on-line sample pretreatment and concentration as opposed to analytical methods where sample pretreatment and concentration are performed off-line from the instrumental set-up. The former includes flow-injection analysis (FIA) and hydride generation (H) methods coupled to cryogenic trapping (CT), the latter covers all classical gas (GC) or liquid chromatography (LC) techniques coupled to a wide variety of detectors 12'13. Hyphenated techniques using on-line sample preconcentration
F/owinjectionanalysis FIA has never been primarily applied to speciation analysis. Speciation analysis with this technique should rather be thought of as a differential determination method using on-line discriminating chemical reactions rather than being based oll true chromatographic separation properties. The sensitivity required for speciation analysis is obtained by direct selective preconcentration on microcolumns, low contamination, and the possibility of on-line matrix pretreatment leading to an overall excellent reproducibility (0.5-1%). Analytes are concentrated from milliliters of the sample to the few microliters which are injected into the detector, thereby allowing concentration factors of 50 to 100. This preconcentration ability can be illustrated with the combination of on-line sorbent extraction/preconcentration procedures applied to the difficult discrete GFAAS detection 14. Speciation conditions can be achieved by several continuous separation techniques such as dialysis, gas diffusion, ion exchange, liquid-liquid extraction or reaction with redox reagents_15 . The high affinity of amphoteric activated alumina can be used to separate oxyanions or cations undel acidic or basic conditions respectively (Fig. 4) 16. Detectors adapted to flow injection techniques in trace metal speciation have to match the continuous flow of the technique. Flame atomic absorption spectrometry (FAAS) and an inductively coupled plasma connected to atomic emission speo
20
trend~" in analytical chemistry, vol. 11, no. l, 1992
TABLE 1. Main hyphenated techniques using chromatography interfaced with atomic spectrometry and applications (adapted from ref. 8) Species
Element
Hyphenated technique Air
Water Sediment
Biol. tissues
Se(IV)/Se(VI), MethylMethylMethylInorg. Se Se(IV)/Se(VI) Se(IV)/Se(Vl)
Se
H-CT-GC-QFAAS GC-QFAAS GC-GFAAS GC-MIP-AES LC-GFAAS HPLC-ICP-AES
+ +
+ +
+
MethylHg(ll) MethylMethyl-
Hg
GC-CVAAS GC-GFAAS GC-MIP-AES HPLC-CVAAS
+
As(III)/As(V), MethylAs(III)/As(V), As(III)/As(V), As(III)/As(V),
Methyl-
As
MethylMethyl-, PhenylMethyl, ASB,ASC
H-CT-GC-QFAAS GC-QFAAS GC-FPD HPLC-GFAAS
I
e0
I'", Cr(lll) ii
I ~
11
I
"
'1
O
e
._c
8O
, I
C
.9
•g
i_ 4o
I
,
J
"J
g "~-g
'' it I
•
.~
Ill I I
[ 11
%111 I
.,Jl,i I i I
Iih.Ii
I
II
I
0
I
I
20
40
Itlllllllll
I
60 Time (s)
+
+
III Cr (VI)
=
+
+
GC-FAAS GC-GFAAS GC-QFAAS HPLC-FAAS HPLC-GFAAS HPLC-MIP-AES HPLC-ICP-AES
•
+ +
+
Pb
v
+
+
Methyl-, Methylethyl-, Ethyl-, Methyl-, Methylethyl-, EthylMethyl-, Methylethyl-, EthylMethyl-, EthylMethyl-, Methylethyl-, EthylMethyl-, Methylethyl-, EthylMethyl-, Methylethyl-, Ethyl
~12 0
+
+
+
H-CT-GC-QFAAS
~160
Blood, flour, leaves + +
+
Sb
200
Standards
H-CT-GC-QFAAS GC-MIP-AES HPLC-H-QFAAS HPLC-GFAAS HPLC-ICP-AES
Sb(lll)/Sb(V), Methyl-
Sn Inorg. Sn, Methyl-, ButylInorg. Sn, Methyl-, Butyl-, PhenylInorg. Sn, Methyl-, Butyl-, PhenylButyl-, Phenyl-
+
8'0
I o Jl I
100
Fig. 4. Speciation of chromium Cr(III)/(VI) by FIA-ICP-AES (emission 267.72 nm). Separation after selective preconcentration and elution from activated alumina microcolumn.
Cr(III)--~Cr(VI) solution, 0.1 mg/I. (From ref. 16.)
Comments
+ +
+
+
+
+
+ + +
+
+ + +
Urine, Sweat
Standards Soils Oil shale, process water
Aerosols Gasoline Gasoline Street dust, aerosols Gasoline Standards Industrial effluent Gasoline, standards
+ + +
Organotin dust
trometry (ICP-AES) have been extensively used. Flow injection methods have been reported for the differentiation of redox species such as Cr(III)/Cr(VI), Fe(II)/Fe(III), As(III)/As(V), Se(IV)/Se(VI). Speciation between inorganic and methyl mercury can also be obtained by the differential affinity of sulphydryl cotton to methyl mercury (Fig. 5) 16. The application of these methods has been mainly directed to the determination of redox species in waters, and sometime in sediments and soil extracts. They are however always limited by potential saturation of the active sites by major ions contained in the matrix. A judicious selection of column packing is therefore needed. Flow injection as a speciation tool is still in its infancy. Among the important future contributions is the possibility of coupling flow injection to highperformance liquid chromatography (HPLC) for on-line sample clean-up, precolumn derivatiza-
21
trends in analytical chemistry, vol. 11, no. 1, 1992
o
E
U "1"
"~
Inorgaric Hg
~
MeHg
t
~
J ~ Time
During the first step, analytes are extracted from the sample matrix by hydride generation under acidic or basic conditions. Hydride generation is now well established for elements such as As, Bi, Sb, Se, Sn, Ge and Te, and it is also efficient for most light alkylated species. In general, the comparison between the pH and pK a of the species shows that the reduction is generally performed at a pH that is a few units below the pK a of the species of interest 2°. The general reaction can be described for alkylated tin compounds21:
i
RxSn(4-x) + NaBH4, H + H+ ~ RxSnH(4-x) + H2 Fig. 5. Reproducibility of FIA-AFS for the speciation of inorganic mercury and methyl mercury with selective preconcentration and elution from a sulphydryl cotton microcolumn. Hg(ll)/MeHg ÷ solution 2 ng/I. (Courtesy C. W. McLeod & W. Jian, Sheffield Polytechnic, UK, presented at the 1990 Winter Conference on Plasma Spectrochemistry, St. Petersburg, USA, January, 1990.)
tion, and the elimination of matrix effects 17. Possibilities of interfacing flow injection to H - C T - G C - Q F A A S also exist and need to be explored. In general, automation of flow injection hyphenated systems will allow higher sample thoughput. Also, if species preconcentration of the sample in the field and stable storage on the same solid support could be achieved under good conditions and later directly eluted in a flow injection hyphenated system, this would change the general prospect of environmental speciation analysis by reducing sample size and analytical errors associated with sample contamination through handling. Preliminary results have been obtained for Cr preconcentration and later speciation in river waters 18.
Hydride generation, cryogenic trapping, chromatographic separation and atomic spectrometry detection This hyphenated technique was first used in 1975 for the determination of methylated forms of selenium in freshwater environments 19. More recently, due to the simplicity and high sensitivity of the method, it has been extensively applied to the determination of butyltin compounds in the environment. It combines the following basic steps: on-line aqueous sample derivatization, preconcentration by cryofocussing, chromatographic separation, detection by AAS in an electrothermally heated quartz furnace and signal processing.
(1)
where x = 1,2,3; and R is methyl-, ethyl- or butyl-. This technique can also differentiate the redox forms of As, Se and Sb as appropriate pH reaction ranges can be selected. Important species of Hg and Pb cannot be directly analyzed by hydride reduction processes. However, the field of application of this technique has now been extended to Hg and Pb as well as to their alkylated derivatives by the use of a new boron alkylating reagent in water (NaBEt4) 22. Hg and Pb were quantitatively ethylated at pH 5 in water according to the following reactions:
Pb2++2
, EtzPb n + . . .
(2)
2 Et2Pb n
~Et4Pb Iv + Pb °
(3)
Me2Pb 2+ + NaBEt 4
~Me2Et2Pb + . . .
(4)
Hg 2 + 2 NaBEt 4
, Et2Hg + . . .
(5)
NaBEt 4
The application of this new derivatization is currently under development. It has been shown to be less sensitive to interferences than hydride generation for the determination of butyltin compounds in complex sediment matrices. The on-line preconcentration and separation steps of derivatized species is one of the major advantages of the H - C T - G C - Q F A A S method. Cryofocussing allows preconcentration factors of 50- to 100-fold and is performed on a small packed chromatographic column coated with a non-polar methylsilicon phase immersed in liquid nitrogen. Separation is achieved upon warming of the trap and species are eluted on the basis of their boiling points as well as their chromatographic properties. This provides a limited separation potential of approximately 3000 plates. The combination of this technique with the highly specific detection cap-
22
trends in analytical chemistrv, vol. II, no. 1. 1992
abilities of AAS using deuterium background correction allows selective recording of inorganic and organometallic forms of the metal present in the sample on the same chromatogram. Fig. 6 presents an extreme effect obtained with sample clean-up of a heavily polluted sediment leachate. Fig. 6a shows the standard chromatogram of the sediment after direct leaching with acetic acid. Fig. 6b displays the increased recovery obtained after clean-up of the leachate. Butyltin compounds are significantly enhanced. Clean-up of the sample also provided evidence of the presence of methylated tin in the sample. The interfacing of the different hyphenation stages is simple, but the system must be able to withstand the high pressure obtained during the hydride generation reaction. Despite its apparent simplicity, this set-up needs to be fine-tuned to obtain high sensitivity. In general, reproducibility with such systems ranges between 5 and 15% (n = 5). Careful automation of each analytical stage should considerably reduce the relative standard deviation routinely obtained. Minimal dead volume is essential for high sensitivity and low tailing of the chromatographic peaks. The continuous gaseous effluent from the chromatographic column is readily atomized in a quartz cell aligned in the beam of an atomic spectrophotometer. Both flame and electrically heated cells are adequate. The addition of H 2 and 0 2 gas in the quartz furnace is critical in obtaining enhanced sensitivity and atomisation mechanisms are based on OH ° free radical processes. This step needs to be developed further in order to achieve maximum sensitivity
m ÷
a
&
=
b ÷
==
£ +
7~ £ ,.t:l ,<
lime ( m i n )
Time (rain)
Fig. 6. (a) Organotin speciation in sediments by H-CT-GCQFAAS after acetic acid (96%) leach. (b) The effect of sample clean-up by a Sep Pak C18microcolumn after conditioning by methanol and acetic acid (96%). (O. F. X. Donard and F. Martin, unpublished data.)
and to control interferences associated with atomisation processes. This inexpensive atomizer is very popular, has seen many designs, and has been applied to several organometallic species of Sn, Se, As, Sb, Pb, and Hg. Inorganic and methylated germanium species can not be dissociated at the low temperature of the QFAAS (1000°C) and are detected by continuous firing of the graphite furnace. Other detectors, such as flame photometric detectors (FPD), provide excellent sensitivity with this set-up but are not selective enough. Atomic fluorescence spectroscopy (AFS) is the preferred detector for mercury analysis. It is more sensitive than AAS and detection limits are at the picogram level for the speciation of inorganic and methylated forms of mercury in the environment 23.
Hyphenated techniques using off-line sample preconcentration Separation by gas chromatography GC has been interfaced successfully with a wide range of detectors. Absolute detection limits obtained with these systems are similar to those using on-line preconcentration since they ultimately use an atomic absorption detector. However, the important difference lies in the initial sample volume. With on-line hyphenated systems, the sample volume can be as large as several hundred milliters if required. With off-line hyphenated systems, only microliter volumes can be injected in the gas chromatograph and therefore preconcentration of the analytes in the aliquot injected is required. Extraction from the sample and preconcentration then needs to be performed off-line in a solvent medium with or without chelating agents. Difficulties exist in the quantitative liquid-liquid extraction of all ionic inorganic and organometallic species. After extraction from the sample, there are several procedures to derivatize and concentrate the analytes prior to chromatographic separation. These procedures include numerous steps. Organometallic compounds can be extracted from the matrix and derivatized by hydride generation. Cryofocussing on glass beads after hydride generation and subsequent GC separation gives the necessary preconcentration required to obtain good sensitivity. In most cases, organolead or organotin compounds react with a Grignard reagent to form volatile tetraalkyl substitutes suitable for chromatography.
23
trends in analytical chemistry, voL 11, no. 1, 1992
Rx(Sn,Pb)(4_x)+ R'MgX Rx(Sn,Pb)R'(4-x)
(6)
solvent
where x = 1, 2, 3; and R is inorg. Sn, methyl-, ethyl-, butyl-, or phenyl. For lead R' can be a propyl-, butyl-, or phenylgroup. For tin, a larger variety of R' substituents have been used such as methyl-, butyl-, pentyl-, and hexyl-groups. After derivatization, the excess Grignard reagent is removed by addition of sulphuric acid and use of a drying agent such as anhydrous CaCl 2. Finally, the samples are concentrated under a gentle stream of nitrogen. These numerous procedures preconcentrate organometallic compounds from large samples thus bringing them into the dynamic range required for speciation studies. The detection limits are in the ng/1 or ng/g range and are suitable for most environmental samples. An important drawback of the technique is the long and tedious off-line extraction/derivatization/preconcentration procedures which, in addition, may be a source of error due to the loss of analytes or of the internal standard. Simultaneous derivatization/liquid-liquid extraction has been applied successfully to minimize these numerous sample pretreatment steps. Recently, on-column derivatization has also been developed. Pellets of NaBH 4 or NaBEt 4 are added on top of the packed column in the injection port. Direct injection of the analytes onto the top of the column results in quantitative hydritization or ethylation of the organometallic species 24'25. Organometallic compounds are then separated on classical chromatographic systems. Interfacing is a critical step in achieving good separation. Transfer lines from the GC to the detectors should be as short as possible, and they should be heated to avoid analyte condensation and decomposition. High-resolution capillary gas chromatography (HRCGC) has probably indirectly increased the sensitivity of the set-up. Increased separation of compounds likely to coelute with organometallic species prevents their competition later in the atomisation and detection processes. The sharp peaks obtained make a heavy demand on the detector's speed-of-data acquisition capacities. Several types of detectors have been coupled to GC for trace speciation. In most cases, the solvent is a potential problem for the detectors used. The most popular detector to date is certainly the electrothermally heated quartz atomizer. Although not highly specific, FPD detectors fitted with a 610 filter to monitor the molecular emission of the
S n - H in nitrogen/hydrogen diffusion flames are gaining increasing popularity, particularly when coupled to capillary GC for the detection of organotin compounds. AES has also been widely used on simple solution matrix samples. AES has a larger dynamic range than AAS. The high cost of the multi-elemental plasma emission detector has limited its use as a chromatographic detector. The increased use of the helium TM010 microwave cavity, developed by Beenakker to operate at atmospheric pressure, has allowed simple interfacing with GC [GC-microwave induced plasma(MIP)-AES]. This plasma source requires low energy and generates high electron temperatures, giving high spectral intensity. It is used for AES of a wide number of elements such as N, P, S, C, Br, CI, H, Si, Hg, Sn and Pb. Detection limits are in the picogram range. It has been basically used for the speciation of alkylated lead and mercury compounds in the environment. Atomic fluorescence is another mode of detection worth mentioning although seldom used. It can provide multi-elemental determination 26. Mass spectrometry (MS) connected to GC is often used for confirmation of the occurrence of new organometallic species found in the environment. The sensitivity of small bench-top systems is too low (ng level) to be used routinely in the full scan mode. However, when used in the selected ion monitoring mode (SIM), detection limits can be improved by 100- to 1000-fold. The continuous improvement in sensitivity of quadrupole instrumentation will certainly allow it to be increasingly used in environmental speciation analysis.
Separationby liquid chromatography Liquid chromatography (LC), at present mainly HPLC, is another popular method for the separation of metallic species prior to detection. This trend does not compete with the other hyphenated techniques mentioned above but rather expands the type of chemical and physical species concerned in speciation analysis. It offers several advantages compared to GC techniques. HPLC methods allow the separation of species without derivatization. Furthermore, the wide variety of chromatographies (adsorption, ion-exchange, gel permeation, normal and reversed-phase chromatography) allows the separation of ions, volatile species, high molecular weight organometallics as well as complex, large-size biological species. Important efforts have been focused on the speciation of arsenic compounds in soil extracts and
24
biological tissues. The polarity of inorganic and most organo-arsenic species makes them amenable to both ion-exchange and reversed-phase HPLC separation. The forms of arsenic concerned are arsenite, arsenate, monomethylarsonate (MMA), dimethylarsinate (DMA), arsenobetaine (ASB), arsenocholine (ASC) and arseno-sugars. The last three species are not volatilized by hydride generation. Organotin and organomercury compounds can also be separated with HPLC methods. The possibility of volatilization by online hydride generation after HPLC separation has already been explored for mercury and tin 27. HPLC coupled techniques are mainly used for the speciation of organometallic or metal-organic molecules in soils, oils or biological tissues after the preliminary clean-up and concentration steps. LC as a separatory tool has also been applied to a wider range of metal-organic compounds such as metal-amino acid complexes or protein-bound metals in biological fluids28'29. Separation of metal chelates and simultaneous detection by AES is also possible 3°. Hyphenation between LC methods and atomic spectrometry detection mostly results in low sensitivity instrumentation. Cryogenic trapping methods are 1000-fold more sensitive than HPLC based techniques. The interface is one of the conditional factors for sensitivity. Due to low efficiency in the nebulization and desolvation of the mobile phase, the major problem is the conversion of analytes to volatile forms which are easily detectable. Another problem lies in matching the eluent flow with the aspiration rate of the nebulizer. Finally, the organic solvent is usually a source of interference in the detector. FAAS was the first candidate for coupling. The low sensitivity of this technique has led several researchers to use GFAAS because of the latter's excellent detection limits. The difficulty in interfacing here lies in the compatibility between the continuous elution of the mobile phase and discontinuous nature of graphite atomisation detection. HPLC-GFAAS systems were developed for the speciation of organometallic compounds of arsenic and tin (Fig. 7) 31'32. Eluents from reversedphase columns may induce a high background molecular absorption requiring the use of the Zeeman effect correction rather than the deuteriumlamp correction. Speciation by LC coupled to plasma systems is 20 times more sensitive than with FAAS and was first demonstrated more than a decade agO33. I C P AES has proven to be relatively interference-free
trends in analytical chernistry, vol. 11, no. 1, 1992
'A.u.) 375
b
250
d
0
aLhl ,ll,
....
IIII kJ
lo 2'o 3o
h,
Time (min)
Fig. 7. Speciation of butyltin compounds by on-line HPLCGFAAS. a = tetrabutyltin (100 mg/I), b = dibutyltin dichloride (400 mg/I), c = tributyltin chloride (400 mg/I), d = monobutyltin trichloride (400 mg/I). Separation on a 250 × 4 mm I.D. Nucleosil 5 CN column. (Courtesy M. Astruc, A. Astruc, R. Pinel, Universit6 de Pau, France.)
for the monitoring of arsenic species and is widely used. Despite their lower sensitivity in comparison to ICP-AES detectors, direct current plasma jets (DCP) are less affected by the solvent and are gaining increasing popularity as LC detectors. A further development has been the coupling of HPLC systems to ICP-MS. Some of the first interfaces realized allowed the determination of meth-
4
5
I
Time4 {rain }
8
Fig. 8. Speciation of arsenic compounds by H P L C - I C P - M S ) . 1 = arsenocholine (31 #g/I), 2 = arsenobetaine (37/~g/I), 3 = As(Ill) (76/tg/I), 4 = dimethylarsinic acid (47/~g/I), 5 = monomethylarsonic acid (41/~g/I), 6 = As(V) (65/~g/I). Separation was performed on an Hamilton PRPX 100 column (250 mm x 4 mm I.D.) Injection loop: 20/A. (Courtesy A. Lamotte, C. Demesmay, M. Olle, Service Central d'Analyse CNRS, Lyon, France.)
25
trends in analytical chemistry, vol. I1, no. 1, 1992
ylmercury34 or dimethylarsinic acid and arsenobetaine 35 in fish extracts. By choosing appropriate chromatographic conditions, extensive speciation of arsenic compounds can be obtained (Fig. 8). Progress in this field can certainly be expected from the coupling of the microbore HPLC system with microconcentric nebulisers and the highly sensitive multielement ICP-MS detector. Future trends
If speciation analysis were only restricted to separation and sensitivity, the merging of new techniques would considerably extend its potential. The combination between supercritical extraction and separation (supercritical fluid chromatography, SFC) techniques would extend separation capabilities in areas where neither GC nor HPLC may be suitable. Detectors would greatly benefit from the direct introduction ef the sample in gaseous form. Supercritical fluid transport to the ICP allows sample introduction with almost 100% efficiency. Interface design is one of the most critical areas where progress is needed to improve sensitivity of detectors coupled to liquid separation techniques. The progress made with ICP-MS detectors is an indication of one of the major future trends of development in speciation analysis. It offers a new range of sensitivity that needs to be explored by tandem association with either GC or HPLC. Ultimate detection limits can also be obtained with laser-enhanced ionization in GC or LC effluents. Laser-enhanced ionization as an element specific detector of GC effluents using an excimer laser and a time-of-flight mass detector can reach absolute detection limits in the attogram (10 -18 g) range. Ion-trap mass spectrometry has also been successfully interfaced with separation techniques such as GC, LC and SFC. It then performs like any other mass spectrometer but provides higher sensitivity. When independent from the coupling and under optimum operating conditions, detection limits are also in the attomole range. The mass range for molecular weight determination is large and there are capabilities for mixture analysis and structural elucidation. The historical development of speciation analysis can be summarized by considering the improvement in determining organometallic molecules of increasing size in water samples and large metalorganic biopolymers in biological systems. Capillary electrophoresis is now applied to the speciation of organometallic forms of arsenic (Fig. 9). It can also be coupled to MS detection. The in-
As203
DMA
-t
As20s MMA
/ a,
1
"
Time (min)
Fig. 9. Speciation of arsenic compounds by Free Solution Capillary Electrophoresis. Detection by UV at 190 nm. Sample concentration, 100 mg/I, injection volume 9 nl. (Courtesy M. Leroy, EHICS, Strasbourg, France.)
creasing use of LC-MS will yield new information on the identity of large metal-organic molecular interactions (e.g. identification of porphyrins, large metal and organometallic complexes). Recently, electrospray interfacing for coupling ionexchange chromatography to mass spectrometry (MS) has been reported to yield effective separation and good sensitivity for the determination of arsenobetaine in dog fish muscle 36. These applications will also benefit from the ongoing developments of heavier instrumentation in organic MS ~'. However, this instrumentation is expensive and will not be easily accessible to many laboratories concerned with environmental problems. One of the main reasons for the slow development in metal speciation studies is the lack of available commercial instrumentation. On a simple basis of cost and sample throughput, refining the integration of FIA in existing hybrid techniques represents another more affordable trend. FIA will allow on-line sample preconcentration and pretreatment. It will also facilitate post-column reactions, such as the addition of matrix modifiers prior to detection, on existing hyphenated systems. Atomic absorption will for some time still be the detector of choice owing to its low cost, high specificity, sensitivity with a quartz electrothermal atomizer cell and wide range of interfacing modes. GC coupled to AES with a microwave induced plasma is now reaching its full range of applications and is commercially available.
trendsin analyticalchernistry, vol. 11, no. 1, 1992
26
in general, high levels of automation are required since the essence of speciation analysis is based on the consecutive yield of physical-chemical reactions leading to quantitation of the analytes. However, it should be borne in mind that speciation analysis requires a strong analytical chain prior to determination. Tandem association is certainly a field that can be instrumentally well mastered. Nevertheless, sample collection, sample concentration and pretreatment are unavoidable steps that needs to be carefully controlled to obtain reliable analyses. Much effort still has to be invested in these preliminary steps. Speciation is now mainly devoted to environmental analysis. Major progress can be expected in other areas such as water treatment, the pharmaceutical industry, biotechnology and medicine.
Acknowledgements The authors would like to thank H. Budzinski, P. Garrigues, F. Fenter and C. McLeod for their help with the manuscript. C. McLeod, M. Astruc, A. Lamotte and M. Leroy are gratefully acknowledged for their contribution.
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14 Z. Fang, M. Sperling and B. Welz, J. Anal. At. Spectrom., 5 (1990) 639. 15 M. Valcarcel and M. D. Luque de Castro, Flow injection Analysis, Principles and Applications, Ellis Horwood, Chichester, 1987. 16 A.G. Cox, A.J. Cook and C.W. McLeod, Analyst (London), 110 (1985) 331. 17 M. D. Luque de Castro, Mikrochim. Acta, submitted for publication. 18 A. G. Cox and C. W. McLeod, Mikrochim. Acta, submitted for publication. 19 Y. K. Chau, P. T. S. Wong and P. D. Goulden, Anal. Chem., 47 (1975) 2279. 20 M. O. Andreae, in C. S. Wong, E. Boyle, K. W. Bruland, J. D. Burton and E. D. Goldberg (Editors), Trace Metals in Sea Waters, Plenum Press, New York, 1983. 21 O. F. X. Donard, S. Rapsomanikis and J. H. Weber, Anal. Chem., 58 (1986) 772. 22 S. Rapsomanikis, O. F. X. Donard and J. H. Weber, Anal. Chem., 58 (1986) 35. 23 N. Bloom, Can. J. Fish. Aquat. Sci., 46 (1989) 1131. 24 S. Clark, J. Ashby and P. J. Craig, Analyst (London), 112 (1987) 1781. 25 J. Ashby, S. Clark and P. J. Craig, J. Anal. At. Spectrom., 3 (1988) 735. 26 A. D'Ulivo and P. Papoff, J. Anal At. Spectrom., 1 (1986) 479. 27 D. T. Burns, F. Glockling and M. Harriot, Analyst (London), 106 (1981) 921. 28 N. Khan and J. C. Van Loon, J. Liq. Chromatogr., 2 (1979) 23. 29 P. E. Gardiner, P. Braetter, V. E. Neggretti and G. Schulze, Spectrochim. Acta B, 38 (1983) 427. 30 J. C. Van Loon, J. Lichjaw and B. Radziuk, J. Chromatogr., 136 (1977) 301. 31 F. E. Brinckman, K. L. Jewett, W. P. Iverson, K. J. Irgolic, K. C. Ehrhardt and R. A. Stockton, J. Chromatogr., 191 (1980) 31. 32 R. Pinel, M. Z. Benabdallah, A. Astruc, M. Pautin-Gautier and M. Astruc, Analusis, 12 (1984) 344. 33 M. Morita and T. Uehiro and K. Fuwa, Anal. Chem., 52 (1980) 351. 34 D. S. Bushee, Analyst (London), 113 (1988) 1167. 35 D. Beauchemin, K. W. M. Siu, J. W. McLaren and S. S. Berman, J. Anal. At. Spectrom., 4 (1989) 285. 36 K. W. Sui, R. Guevremont, J. C. Y. Leblanc, G. J. Gardner, and S. S. Berman, J. Chromatogr., 554 (1991) 27. 37 M. Linscheid, Fresenius' J. Anal. Chem., 337 (1990) 648.
i,( ~ X. Donard is a CNRS research scientist at th~Laboratoire de~Pt~6~f~hysique et Photochimie Molecu/air~ at the University of Bordeaux (France)J His interests are in analytical chemistry applied to chemida/ oceanography. He develops analytical methods by interfacing gas chromatography techniques to atomic spectrometry. Fabienoe M. Martin is a PhD student in Chemical Physics applied to the Environment in the same laboratory. She is doing a PhD in analytical chemistry on the hyphenation between gas chromatography and atomic spectrometry detectors such as atomic absorption or inductively coupled plasma-mass spectrometry.