JOURNAL O F ANALYTICAL ATOMIC SPECTROMETRY NOVEMBER 1994 VOL. 9 1279 Speciation of Inorganic Mercury(i1) and Methylmercury by Vesicle-mediated High-performance Liquid Chromatography Coupled to Cold Vapour Atomic Absorption Spectrometry 6. Aizpun Maria Luisa Fernandez E. Blanco and Alfred0 Sanz-Medel* Department of Physical and Analytical Chemistry University of Oviedo Julian Claveria 8 33006-Oviedo Spain A novel high-performance liquid chromatography (HPLC) separation coupled to cold vapour atomic absorption spectrometry (CVAAS) detection for the speciation of inorganic mercury(i1) and methylmercury is described. The mercury species can be successfully separated within 8 min by using a vesicular mobile phase of didodecyldimethylammonium bromide (DDAB) in acetate buffer containing 5% v/v of modifier methyl cyanide (acetonitrile) and 0.005% v/v of 2-mercaptoethanol.The stationary phase is a &-bonded silica column previously modified by the passing of a solution of DDAB. Detection limits of 0.1-0.2 pg I-' of mercury were achieved after off-line preconcentration of the aqueous samples using C Sep-pack cartridges modified with 2-mercaptoethanol solutions. This vesicle-enhanced HPLC-CVAAS approach has been applied to the speciation of inorganic mercury(i1) and methylmercury in spiked sea-water and human urine. Recoveries obtained ranged between 91 -1 03% both for the inorganic and organic mercury species. Keywords Vesicles; high-performance liquid chromatography; cold vapour atomic absorption spectrometry; mercury speciation and preconcentration ; sea-water and human urine It is now well known that the toxicological and biological effects of trace elements depend on their chemical forms in the sample.' In particular inorganic mercury is converted into the much more toxic methylmercury compound in the environment by a number of biological processes. The latter form is of particular concern because of its enhanced toxicity lipophilic- ity bioaccumulation and volatility compared with inorganic mercury. These facts assumed special importance following earlier pollution incidents such as that at Minamata Bay in Japan and were decisive in realizing that analytical measure- ments of the more toxic forms would be more meaningful than total element determination.2 A number of methods have been applied to the determination of mercury species in different matrices.The strategies most widely used involve gas chromatography (GC) separation with different types of detectors including the electron capture detector ( ECD);3 atomic absorption spectrometry (AAS);4 and microwave-induced plasma atomic emission spectrometry (MIP-AES).' More recently mercury speciation has been also carried out by coupling high-performance liquid chromatogra- phy (HPLC) with specific detectors such as AAS,6-1' induc- tively coupled plasma atomic emission spectrometry (ICP- AES)" and ICP-mass spectrometry ( ICP-MS).13,'4 Although the ICP-AES and ICP-MS detectors have unique analytical capabilities for speciation their high instrumental and running costs make them more difficult to be adopted widely as common chromatographic detectors.Conventional nebuliz- ation AAS is the most popular specific detection system,15 owing to its simplicity inexpensive instrumentation and ready availability. The sensitivity of this detector in its conventional use is however insufficient for the speciation of very low concentrations of mercury species in some real samples (e.g. natural water samples). Therefore high sensitivity sample introduction techniques [e.g. post-column derivatization of mercury species to form a cold vapour (CV)] and perhaps preliminary preconcentration have to be used to improve the analytical sensitivity of the HPLC-AAS Reversed-phase liquid chromatography using 2-mercapto- ethan~l'~~'~-'* (or other molecules with the -SH in an aqua-organic mobile phase have been proposed for separation.However the introduction of these mobile phases containing relatively high percentages of organic * To whom correspondence should be addressed. solvents into plasmas results usually in a decrease in sensi- tivity higher plasma background increased instability and even eventual extinction of the plasma.'' Therefore the use of alternative HPLC mobile phases which do not use organic solvents such as in micellar liquid chromatography (MLC) could be advantageous.20 In fact MLC has shown some important advantages including enhanced selectivity versa- tility rapid gradient elution capability low toxicity low cost and the ability to simultaneously chromatograph both hydro- philic and hydrophobic solutes.21 Unfortunately MLC also suffers from some drawbacks including loss of efficiency22 and solvent strength.23 Although MLC has been extensively studied for separations during the last years no attention has been paid to the use of mobile phase systems containing other surfactant-based organized assemblies such as vesicles.Very recently it has been established that HPLC separation followed by HG-ICP-AES detection can be a synergic combination uia the use of di- dodecyldimethylammonium bromide (DDAB) vesicles as mobile phases for the speciation of toxic arsenic species.24 In this paper the new strategy of vesicle-mediated HPLC separations is coupled to vesicle-enhanced CVAAS detection as it has been shown that CV generation of mercury can be improved in DDAB vesicles.z5 This vesicular HPLC-CVAAS technique is applied to mercury speciation in sea-water and human urine samples after adequate preconcentration in c18 cartridges modified with 2-mercaptoethanol.Experimental Apparatus A Knauer Model 6400 HPLC pump with an attached sample injection valve equipped with a 100mm3 loop were used for eluent delivery and sample introduction. The analytical column was a Spherisorb ODS 2 (250x4.6mm id.) packed with 10 pm C18-bonded silica stationary phase previously modified by passing DDAB solution as described below. A four channel peristaltic pump HP4 Minipuls 2 Gilson and a laboratory-made gas-liquid separatorz6 constituted the con- tinuous CV generator. A schematic diagram of the whole HPLC-CVAAS system is presented in Fig. 1. An ultrasonic device from Sonics and Materials Model VC (250 W) was used for DDAB vesicle preparation.1280 JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY NOVEMBER 1994 VOL.9 mobile phase Pump Hydrochloric acid (1%) Fig. 1 Schematic diagram of the coupled HPLC-CVAAS system for mercury speciation C,* column A Unicam Model PU 9400X atomic absorption spec- trometer equipped with a T shaped absorption quartz cell (8 mm i.d. 12 cm length) was used for absorption measure- ments of mercury vapour at 253.7 nm. Reagents Methylmercury stock solution (100 mg I-' of Hg) was obtained by dissolving the appropriate amount of methylmercury chlor- ide salt (Merck) in 10cm3 of acetone which was made up to 100 cm3 with ultrapure Milli-Q water. This stock solution was stored in a glass bottle at 4 "C.Inorganic mercury was obtained from Merck as a 1000 mg dm-3 Hg solution. Working standard solutions of both mercury compounds were freshly prepared daily by diluting the stock solutions with ultrapure Milli-Q water. The DDAB vesicular solution (lo-' mol dm-3) was pre- pared by dissolving 0.4626 g of DDAB (Fluka) in 100 cm3 of Milli-Q water and sonicating this solution (with a power output of 60 W) for 10 min. Sodium tetrahydroborate solution (1 YO m/v) was prepared by dissolving 1 g of NaBH ( Probus Barcelona) in 100 cm3 of 0.1 % m/v NaOH solution. Filtration of the solution through a Whatman grade 4 filter paper before use was carried out. This solution was stored at 4 "C and prepared weekly. Hydrochloric acid solution (1% v/v) was prepared from concentrated HCl (Merck) and Milli-Q water.The mercury- selective complexing agent 2-mercaptoethanol was obtained from Merck and used without further purification. The HPLC grade methanol and methyl cyanide (acetonitrile; Romile Chemicals) were used. All other chemicals were of analytical-reagent grade and distilled and de-ionized (Milli-Q system Millipore) water was used throughout. Procedures HPLC column modification The C bonded silica reversed-phase column was modified by passing a total of 500 cm3 of a DDAB (1 x mol dm-3) aqueous solution in 50% methanol at a flow rate of 1 cm3 min-'. Milli-Q water was then passed through the column for 30 min at the same flow rate. This modified column was kept in water when not in use. Mercury speciation Vesicular mobile phases were prepared by dissolving the appropriate amount of DDAB in water containing 0.005% v/v of 2-mercapthoetanol 5% v/v acetonitrile and buffered with ammonia acetate (0.01 mol dmP3) at the desired pH.The mobile phase was degassed by ultrasonicating for 30 min prior to use. This mobile phase was then continuously pumped through the analytical column at a flow rate of 1.5 cm3 min-' and 100mm3 of the working solutions of the mercury com- pounds were injected for analysis. The eluent at the exit of the HPLC column was first mixed with a 1% v/v HC1 solution and then mixed with the 1% m/v NaBH solution for the mercury CV generation which was continuously swept through the gas-liquid separator to the T quartz cell of the atomic absorption spectrometer by a stream of argon (250 cm3 min-I) see Fig.1. All separations were performed at room temperature under isocratic conditions. Each separation was attempted under several different combinations of organic modifier DDAB vesicle concentrations pH etc. The best chromatographic resolution of the various set of conditions tested was obtained using the separation conditions summarized in Table 1. Optimum experimental conditions finally selected for CV generation and AAS detection after preliminary investigations are also summarized in Table 1. Peak heights from the chroma- tograms were used in all mercury quantifications. Sample collection and pre-treatment Sea-water samples were collected in a Spanish coastal region (Gijon) of the Cantabric Sea and immediately acidified (by the addition of ultrapure nitric acid to have a final pH of 2) for storage in pre-cleaned polypropylene bottles.The samples were filtered through a Millipore 0.45 pm membrane. The pH was adjusted to 7 with diluted ammonia before the speciation analysis. Human urine samples were filtered through a Millipore 0.45 pm membrane. Preconcentration step Preconcentration of mercury compounds from water and urine samples was carried out using Sep-pack C18 [trichloro(octa- decyl)silane chemically bonded to Porasil A] cartridges modi- fied with 2-mercaptoethanol solution as described below. Cartridges were activated by washing with 7cm3 of meth- anol which was subsequently displaced with 7 cm3 of ultrapure water. Then the cartridge was modified by passing through it 10 cm3 of 0.5% v/v 2-mercaptoethanol aqueous solution.Volumes of 100cm3 of the samples (standard solutions sea- water and human urine) were then pumped through the modified cartridge by using a Gilson Minipuls 2 peristaltic pump at a flow rate of 10 cm3 min-'. The mercury species Table 1 HPLC-CVAAS Experimental conditions for the mercury speciation by Chromatography Column Temperature Sample volume Mobile phase Flow rate NaBH cv HCI Ar carrier Wavelength Lamp current Slit-width AAS C,,-bonded silica 10 pm particle size 250 x 4.6 mm i.d. (modified with 1 x rnol dmT3 DDAB) Room temperature 100 mm3 10 mmol dm-3 ammonium acetate buffer + 2-mercaptoethanol+ 1.5 cm3 min-l 5% acetonitrile +0.005°/a 2 x mol dm-3 vesicle of DDAB pH=5 1% m/v NaBH (in 0.1% NaOH) flow rate 1% v/v flow rate 1 cm3 min-' Flow rate 250 cm3 min-' lcm3 min-' 253.7 nm 6 mA 0.5 nmJOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY NOVEMBER 1994 VOL.9 1281 loaded in the cartridge were eluted with 10 cm3 of acetonitrile. These acetonitrile solutions were rotary evaporated and the residue was dissolved in 1 cm3 of the mobile phase ( x 100 preconcentration factor) and 100 mm3 of this solution were injected for HPLC determinations. Results and Discussion Chromatographic Separation The CI8 column was previously modified with DDAB as detailed above. The DDAB coating formed proved to be very stable and resistant to water and to the mobile phase passing Although organic solutions e.g. methanol could remove the coating the modified column was very durable in the recommended vesicular operation.In fact no significant column behaviour indicating any sort of degradation was observed after several months of daily usage. In order to investigate the effect of the pH of the mobile phase the retention times of both solutes under study were measured in the pH range 4-612,16 using a mobile phase consisting of 0.002 mol dm-3 ammonium acetate 5% v/v acetonitrile 0.005% v/v 2-mercaptoethanol and 2 x mol dm-3 DDAB vesicles. As expected when the pH of the mobile phase was varied from 4 to 6 no significant change of retention time of the neutral mercaptoethanol- mercury complexes12*'6 was observed. A mobile phase of pH 5 was then chosen for further studies. After fixing the pH the effect of increasing buffer concen- trations in the mobile phase on retention times of solutes was investigated.The results obtained (Fig. 2) indicate that reten- tion times tend to decrease as the concentration of ammonium acetate increased up to an ammonium acetate concentration above of 0.01 mol dm-3. At higher buffer concentrations the retention time change was very small Therefore a concen- tration of 0.01 mol dm-3 was chosen. This concentration is considerably lower than that generally used (0.06 mol dm-3) in reversed-phase HPLC with conventional hydro-organic mobile phases.12 The use of such high salt concentrations generated in our system produced irreproducibilities in the measured retention times and very long column equilibration times. The effect of increasing the acetonitrile concentration in the vesicular mobile phase on the separation of the two mercury compounds investigated was also studied.The results were compared with those obtained in the absence of DDAB (using a typical reversed-phase system with an unmodified ClS column). The comparative results observed are given in Table 2 which shows that for both HPLC systems investigated (conven- tional and vesicular) the overall peak broadening and retention 1 I I 1 I 0 0.005 0.01 0.015 0.02 0.025 [Ammonium acetatel/mol I ' Fig.2 Effect of buffer concentration on retention times A Inorganic mercury and B methylmercury. Mobile phase 0.005% 2-mercaptoethanol 5% acetonitrile 2 x lop4 DDAB vesicles in ammonium acetate buffer (pH=5); flow rate 1 cm3 min-'. All other experimental conditions as described in Table 1 times of inorganic mercury and methylmercury were sensitive to the percentage of acetonitrile added to the mobile phase.As expected using conventional reversed-phase HPLC the retention times were reduced by increasing the amount of acetoni trile present. However high percentages of ace toni trile greater than 25% were needed in order to obtain acceptable resolution values for inorganic and organic mercury. As shown in Table 2 acetonitrile addition also decreased the retention times in the vesicular HPLC system but in this case good separations were achieved even in the absence of the organic modifier. This finding can be of great importance for further coupling of the exit of the column to conventional nebulization ICP-AES or ICP-MS detect01-s.~~ In addition when the same amount of organic modifier was used in both systems (see the results for 0-5% acetonitrile in Table 2) lower retention times were obtained for the vesicular chromatography.It should be realized that the surface of the C stationary phase used had been previously modified by the surfactant molecules from the DDAB solution. Therefore a partially coated polar stationary phase will result which allows for not only classical hydrophobic interactions but also for electrostatic interactions with ionizable solutes. These will distribute between the modified stationary phase and the charged vesicles into the mobile phase.23 Considering the nature of the solutes for separation (neutral complexes of mercury with 2-mercaptoethanol) only hydrophobic inter- actions should occur. Therefore the observed decrease in the retention times in the presence of acetonitrile and DDAB could be rationalized by the fact that hydrophobic attractions of the neutral solutes by the surfactant-modified stationary phase are weaker than those observed with the conventional unmodified C18 stationary phase.The dependence of the solute retention time on the surfactant concentration in the mobile phase was evaluated. To do so the influence of using or not using sonication of the surfactant mobile phase (presence and absence of mono-dispersed DDAB vesicles in the mobile phase respectively) as a previous step to the chromatographic separation was investigated. Results obtained in these experiments have been plotted in Fig. 3 and show that the retention time decrease observed with surfactant addition only takes place if mono-dispersed DDAB vesicles are present in the mobile phase (ie.with previous sonication of the surfactant solutions). This effect of vesicles in the separation is stronger for the solute with stronger retention time (inorganic mercury which will form mercury 2- mercaptoethanol neutral species and will be the last species to elute (see Fig. 4). Finally shorter retention times were obtained with increas- ing mobile phase flow rates (resulting in greater peak height) and a 1.5 cm3 min-' flow rate was selected as optimum for the speciation analysis. Fig. 4 illustrates that a mobile phase consisting of 2 x mol dm-3 DDAB vesicles buffered at pH 5 with ammonium acetate (1 mmol dm-3) containing 0.005% v/v mercaptoethanol 5% acetonitrile and delivered at 1.5 cm3 min-l allowed a most adequate isocratic separation of methylmercury and inorganic mercury in an aqueous mixture.Analytical Characteristics of the Method In order to evaluate the precision of this novel chromato- graphic speciation method eight injections (100 mm3) of a standard mixture containing known amounts of the two mer- cury compounds concentration (200 pg dm-3 in the element) were made. The relative standard deviation (RSD%) of the peak height results calculated for inorganic mercury were always worse than those for methylmercury at 20ng of the metal level (Table 3). The observed detection limits (as calcu- lated for a signal-to-noise ratio of three) for an injected volume of 100 mm3 are also included in Table 3 and show values of 10-20 yg dmP3 rather insufficient for the determination of1282 JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY NOVEMBER 1994 VOL.9 I J Table 2 Effect of the percentage of organic modifier in the mobile phase on the retention times (t,) and resolution (Rs) 2 min ~~~ Acetoni trile (%I 25 20 15 10 5 2.5 0 Conventional reversed phase HPLC* tr (Hg)/min 5.30 5.35 5.83 7.50 14.17 14.47 > 30 t (MeHg)/min 6.25 6.33 6.80 7.50 12.45 12.97 > 30 Vesicular HPLCT - Rs tr ( Hg )/min tr (MeHg)/min Rs 0.05 0.84 0.30 0.00 0. so 10.50 7.33 1.50 0.60 12.97 8.43 1.98 -_ 17.40 11.32 1.95 * Column (250 x 4.6 mm i.d.) 10 pm bonded silica; mobile phase 0..005% 2-mercaptoethanol in 0.06 mol dmP3 ammonium acetate buffer t Column (250 x 4.6 mm i.d.) 10 pm c18 bonded silica (modified with lop3 mol dmP3 DDAB); mobile phase 0.005% 2-mercaptoethanol mol dm-3 vesicles of DDAB in 0.01 mol dmP3 ammonium acetate buffer (pH 5) flow rate 1 cm3 min-'. Other experimental conditions (pH 5) flow rate 1 cm3 min-'.Other experimental conditions as described in Table 1. 2 x as described in Table 1. usual levels of methylmercury and inorganic mercury in natural water samples (e.g. sea-water where preconcentration should then be used). Calibration graphs for each of the mercury species were worked out and turned out to be linear over several orders of magnitude ranging from the detection limit to 400 pg dm-3 (maximum concentration tested). I ~ 0 0.5 1 .o 1.5 2.0 2.5 [DDA81/10-'mol I-' Fig. 3 Effect of DDAB concentration in the mobile phase on retention times DDAB vesicles (solid line) and DDAB without sonication (broken line).A Inorganic mercury and B methylmercury. Experimental conditions are given in Table 1 2 Time - Fig. 4 Typical chromatogram of a standard mixture containing 100 pg dm-3 of each mercury specie 1 methylmercury and 2 inorganic mercury. Experimental conditions as described in Table 1 Preconcentration of Mercury Species It is well known that the sulfydryl groups (-SH) exhibit a relatively high affinity for mercury. This fact prompted us to investigate the possibility of increasing the detectability of mercury species in our vesicle-mediated HPLC separation by on-line complexation preconcentration. In this vein Sep-pak cartridges packed with CI8 were modified (impregnated) with the chelating agent 2-mercaptoethanol as described under Procedures.Known amounts of inorganic mercury and methylmercury were added to ultrapure water and preconcentrated on a Sep- pak C cartridge modified by passing through an aqueous solution of 5% v/v of mercaptoethanol. This modified cartridge proved to be adequate for preconcentration of up to 400 ng of both types of solutes (recovery 91 & 5%) and was used for the off-line column extraction-preconcentration of the sought species in sea-water and urine samples. For such samples at the pH around 7 the two species of interest were extracted quantitatively in the modified cartridge and so this pH was used here for further work. Samples (100 cm3) were used in the test for preconcentration in the cartridge with a loading rate of 10 cm3 min-'.The loaded mercury species were then recovered quantitatively from the cartridge for injection in the HPLC column as detailed already under Procedures (Preconcentration step). Analysis of Real Samples The complete analytical procedure (preconcentration and determination by vesicular HPLC-CVAAS) described was applied to the speciation of the two mercury compounds in sea-water. To do that aliquots of acidified sea-water (100 cm3) samples were spiked with concentrations between 0-4 pg dm-3 of inorganic and methyl mercury. The spiked samples were Table 3 Figures of merit of the vesicle-mediated HPLC-CVAAS method (without preconcentration) Mercury species DL/pg dm - RSD YO)^ Linear range/pg dm-3 t,/min Methylmercury 10 3.5 10-400 4.83 Inorganic mercury 16 6.9 16-400 7.15 * Detection limit calculated as 3 times the baseline noise.t Relative standard deviation 8 replicates of each mercury species injected.JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY NOVEMBER 1994 VOL. 9 1283 Table 4 Recoveries of mercury species added to sea-water (100 cm’) DL for methylmercury with 100 1 preconcentration 0.10 pg dm-3; and DL for mercury with 100 1 preconcentration 0.16 pg dm-3 Methylmercury Inorganic mercury Absolute amount Recovery Concentration Absolute amount Absolute amount Recovery added/pg dm - added/ng recovered/ng* (Yo) recovered/ng* (Yo) - 20 - 0 0 0 2 200 177 89 184 92 3 300 272 91 27 1 90 4 400 362 91 359 91 * Hg found in the preconcentrated solution (1 cm3). Table 5 Recoveries of mercury species added to human urine (100 cm3) Methylmercury Concentration Absolute amount Absolute amount added/mg dm-3 added/pg recovered/pg 0 0 0 0.100 10 10.3 0.300 30 28.6 0.003 0.300 0.310* 0.004 0.400 0.405* Inorganic mercury Recovery Absolute amount Recovery (Yo) recovered/pg (%) - 103 95 103* 101* - 11.4 27.0 0.272* 0.371* - 114 90 91* 93 * * After preconcentration procedure. then filtered through a 0.45 pm filter and their pH adjusted to 7 with diluted ammonia to be analysed immediately.Recoveries obtained of the added mercury spikes were always higher than 90% as shown in Table4. Very small amounts of inorganic mercury only (about 0.2 pg dm-3) were detected in the natural sea-water samples used in these recovery experiments. The blank values for each mercury species were estimated by parallel experiments applying the same preconcentration- speciation procedure to 100 cm3 of Milli-Q water Again only inorganic mercury was detected in the blank solution at concentrations of 0.4k0.2 pg dm-3 according to the con- tamination of the reagents (namely 2-mercaptoethanol used without purification).Natural sea-water samples were treated as unknown samples and analysed for inorganic and methyl- mercury following the recommended procedure. The applicability of the proposed vesicle-mediated method for the mercury speciation in human urine was also evaluated. As real samples of the human urine available did not contain detectable mercury and methylmercury they were spiked with concentrations between 0.1-0.3 mg dm-3 of the two mercury compounds. The spiked samples were filtered and analysed for both species without preconcentration.Recovery values for different levels of the two species are shown in Table 5. Parallel experiments were performed with samples of 100 cm3 with low levels of spiked samples which were preconcentrated and analysed following the procedure described above. Recoveries obtained ranged between 91-103% (see Table 5). In brief the recommended strategy offers a reliable analytical method to speciate toxic mercury at very low concentration levels in sea-water and urine which could be extended to other environmental and/or biological samples. Conclusions The coupling of a vesicle-mediated HPLC separation with CVAAS detection of mercury atoms has proved to be a novel rapid and efficient method for the speciation of methylmercury and inorganic mercury(11).These two species can be separated in less than 8 min with DDAB vesicular mobile phases and a DDAB previously modified c18 bonded silica stationary phase. The presence of vesicles in the mobile phase seems to play a role in the neutral mercury species finally formed for HPLC separation and this role could be similar to that described23 for micelles in MLC. This ‘vesicular’ chromatography makes it possible to carry out the separation of the two mercury compounds using very low percentages of organic modifier in the mobile phase as compared with those required in conven- tional reversed-phase chromatography. This means improved detection performance using specific (atomic) detectors particularly using plasmas.27 A new method for the off-line preconcentration of mercury species by solid-liquid extraction with sep-pack cartridges modified with 2-mercaptoethanol solutions is also proposed here.This technique exhibits much higher efficiencies for recovery of mercury compounds than other recent approaches.” The modified c18 material has shown to be an effective solid-phase complexing agent (extractant) which pro- vides virtually quantitative retention of both investigated mer- cury species from sea-water and human urine samples and thus it should be adequate for the on-line preconcentration HPLC-CVAAS speciation of mercury in environmental and biological samples. These vesicle-mediated strategies could be extended to the speciation of other toxic metals of environmental concern,2 particularly those’ using HPLC-plasma detection.28 1 2 3 4 5 6 7 8 9 10 11 12 13 References Robinson J.B. and Tuovinen 0. H. Microbiol. Rev. 1984,48,95. Environmental Analysis Using Chromatography Interfaced With Atomic Spectroscopy Eds. Harrison R. M. and Rapsomanikis S. Ellis Horwood Chicester 1989. Horvart M. Byrne A. R. and May K. Talanta 1990 37 207. Fischer R. Rapsomanikis S. and Andreae M. O. Anal. Chem. 1993 65 763. Bulska E. Emteborg H. Baxter D. C. Frech W. Ellingsen D. and Thomassen Y. Analyst 1992 117 657. Van Loon J. C. Anal. Chem. 1979 51 1139A. Munaf E. Haraguchi H. Ishii D. Takeuchi T. and Goto M. Anal. Chim. Acta 1990 235 399. Fujita M. and Takabatake E. Anal. Chem. 1983 55 454. Lupsina V. Horrat M. Jeran Z. and Stegnar P. Analyst 1992 117 673.Rencede M. C. R. Campos R. C. and Curtis A. J. J. Anal. At. Spectrom. 1993 8 247. Sarzanini C. Saccchero G. 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Blanco Gonzalez E. and Sanz-Medel A. J. Anal. At. Spectrom. 1993 8 815. 25 Sanz-Medel A. Fernandez M. R. ValdCs-Hevia M. C. Aizpun B. and Liu Y . M. Talanta 1993 40 1759. 26 Menendez Garcia A. Sanchez Uria E. and Sanz-Medel A. J. Anal At. Spectrom. 1989 4 581. 27 Aizpun B. Fernandez M. R. and Sanz-Medel A. J. Anal. At. Spectrom. 1993 8 1097. 28 Element Specific Chromatographic Detection by Atomic Emission Spectroscopy Uden P. C. American Chemical Society Washington DC 992. Paper 4/01 724B Received March 23 1994 Accepted June 10 1994