Anal. Chem. 1990, 62, 1161-1166
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Determination of Arsenobetaine, Arsenocholine, and Tetramethylarsonium Cations by Liquid Chromatography-Thermochemical Hydride Generation-Atomic Absorption Spectrometry Jean-Simon Blais,* George-Marie Momplaisir, and William D. Marshall Department of Food Science and Agricultural Chemistry, Macdonald College, 21,111 Lakeshore Road, Ste-Anne de Belleuue, Quebec, Canada H9X 1CO
A novel hlgh-performance liquid chromatography-atomic a b sorption spectrometry (HPLC-AAS) Interface based on thermochemical hydride generatlon has been developed and optlmlzed for the determlnatlon of arsenobetalne [(CH,),As+CH,COOH], arsenochdne [(CH,),As+CH,CH,OH], and tetramethylarsonlum [(CH,),As+] catlons. I n thls quartz Interface, the methanollc HPLC eluent was nebullzed by thermospray effect and pyrolyzed In a methanol/oxygen kinetic flame and the analytes were thermochemlcally derlvatlzed to the hydrlde derlvatlve In the presence of excess hydrogen. The vdatlle derlvative was then transporled to a cod diffusion H2/02flame atomizer. The fact that arsenic pentoxide was also derlvatlzed, and that no slgnal was observed In the absence of postthermospray hydrogen or the absence of cool dlffuslon flame, corroborated the thermochemlcally mediated arslnegeneratlon mechanism. Factorlal models predlctlng the performance of the Interface at dmerent levels of five selected varlabk suggested that (a) both reverse- and normal-phase HPLC eluents were compatible with the Interface and (b) the performance of thls system was relatively lnsenslthre (less than 50% varlatlon In response) to changes over wide ranges In the operating parameters. At concentrations up to 10-fold excess, potential lnterferents [(CH,),S+, (CH,),Pb+] dld not affect, slgnlflcantly, the postulated thermochemical hydride generation (THO) process. However, a 10-fold molar excess of (CH,),Se+ decreased the response of the analyte by 48 % . Virtually Identical responses were observed for equimolar amounts of tetramethylarsonlum, arand dlmethylarslnlc senochollne, arsenobetalne [As( -I I I)], acld [As(III)]. Arsenic pentoxkle [As(V)] was detected with 75 % efflclency, relative to the response of the organoarsenlc compounds. The analytes were separated Isocratlcally In a cyanopropyl bonded phase HPLC column, using a methanollc eluent containing 30% (v/v) diethyl ether, 1% (v/v) acetlc acld, and 0.05% (v/v) triethylamine. The absolute llmlts of detectlon for the arsenobetalne, arsenochollne, and tetramethylarsonlum catlons were 13.3, 14.5, and 7.6 ng, respectlvely. The low purchase and operating costs of this Interface coupled with Its relatlvely low llmlts of detectlon and good reproduclbllity make It a candidate for routlne HPLCAAS monitoring of these arsenic-contalnlng specles.
The discovery of appreciable concentrations of different arsonium compounds [ (CH3)3RAs+X-; R = CH3, CH2CH2CH20H,CH,COOH] in a variety of marine organism (1-7) has created a demand for routine determination of these analytes in commercial fisheries products. Although high-performance
* To whom correspondence should be addressed. 0003-2700/90/0362-1161$02.50/0
liquid chromatography-inductively coupled plasma (HPLCICP) ( 1 , 4 , 5 , 8 ) ,high-performance liquid chromatographyinductively coupled plasma mass spectrometry (HPLC-ICPMS) (7), or high-performance liquid chromatography-graphite furnace atomic absorption spectrometry (HPLC-graphite furnace-AAS) (6,9)instruments have been applied successfully to the determination of certain organoarsonium compounds, their high purchase price and operating cost make it difficult to justify their dedicated use for a specific procedure. Yet routine analyses and systematic biological studies often require a continuous access to a sensitive detection procedure. For arsenic, the electrothermal (10) and cool diffusion flame (11,12) quartz atomizersare several orders of magnitude more sensitive than conventional flame-AAS atomizers. However, these atomizers are limited to arsenic compounds that form volatile hydride derivatives. Different organometallic and inorganic compounds of As(II1) have been speciated by HPLC-hydride generation-quartz tube electrothermal atomization AAS (HPLC-HG-QTAAS) (13). However, most arsonium compounds (R4As+)are not amenable to this on-line liquid-phase hydride generation technique because they do not react with reducing agents. In this paper, a novel HPLC-thermochemical hydride generator-AAS interface for the determination of biogenic arsonium species (arsenobetaine, arsenocholine, and tetramethylarsonium cations) is presented. This on-line interface is based on thermospray nebulization of the HPLC methanolic eluent, pyrolysis of the analyte in a methanol/oxygen flame, gas-phase thermochemical hydride generation (THG) using excess hydrogen, and cool diffusion flame atomization of the resulting arsine in a quartz cell mounted in the AAS optical beam. EXPERIMENTAL SECTION Thermochemical Hydride Generator. Diagrams of the thermochemical hydride generator (THG) main body and of the complete assembly are presented in parts A and B of Figure 1, respectively. The all-quartz (LaSalleScientific,Inc., Guelph, ON) main body (Figure 1A) consisted of an optical tube (a, 9 mm i.d. X 11 mm 0.d. X 12 cm) which was positioned in the AAS optical beam, an analytical flame tube (b, 4 mm i.d. x 6 mm 0.d. X 8 cm); a combustion chamber (c, 7 mm i.d. X 9 mm 0.d. X 3 cm) located within the side arm of the lower T-tube; a thermospray tube (d, 4 mm i.d. X 6 mm 0.d. x 8 cm); and oxygen/hydrogen inlets (e, f, 2 mm i.d. X 3.2 mm 0.d. X 5 cm). The oxygen inlet was located 2.5 cm upstream from the hydrogen inlet. The combustion chamber-thermospray tube assembly met the analytical flame tube at an angle of 45O. The complete assembly (Figure 1B) was composed of the main body, a capillary transfer line (A, 50 pm i.d. X 20 cm deactivated silica tube, Chromatographic Specialties, Brockville, ON) which was connected to the HPLC column outlet; a quartz guide tube (B, 2 mm i.d. X 3.2 mm 0.d. X 10 cm, with outlet bore constricted to 1 mm with a glass blowing torch) which served to center the capillary (A) within the thermospray tube 0 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990
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A
f P
B I1
I I
E---+
HPLC ELUATE
a
Flgure 1. Thermochemkal hydride generation interface: quartz body (A) cmsiSUng of optical tube (a),analytical flame tube (b), combustion chamber (c), thermospray tube (d), oxygen and hydrogen inlets (e-f);
Complete assembly (8)comprising a capillary transfer line (A), quartz guide tube (B), heath element (C), modified Swagelok assemblies (D), analytical oxygen inlet tube (E). (d); and a coil of resistance wire (C, 40 cm of 22-guage Chrome1 875 alloy, Hoskins Alloys, Toronto, ON) which was insulated with refractive wool (Fiberfrax, The Carborundum Co., Niagara Falls, NY) and surrounded by a shaped firebrick casing held together by a screw-clip. Two staidem steel Swagelok assemblies (D)were modified (14)and served to position the guide tube (B) within the thermospray tube (d) and the analyticaloxygen inlet ( E quartz tube, 2 mm i.d. X 3.2 mm 0.d. X 15 cm) within the analytical flame tube (b). The guide tube B was positioned in the thermospray tube 1 cm behind the downstream end of the heating element. The outlet edge of the analytical oxygen inlet E was vitrified by using the glass blowing torch. The tip of the analytical oxygen inlet (E) was immobilized 0.5 cm from the optical tube intersection, so as to maintain the tip of the diffusion flame just below the entrance to the optical tube. Thermospray oxygen and hydrogen were channeled from flowmeters (Matheson, Toronto, ON) by TFE tubing (2.48 mm i.d. X 4 mm o.d., Cole-ParmerCo., Chicago, IL), which were heat shrunk on to the quartz tube gas inlets using a Bunsen burner. The contraction of the TFE tube upon cooling formed a tight seal. The analytical oxygen was introduced in tube E through the modified Swagelok assembly. The optical tube was mounted in an aluminum casing as described elsewhere (14) and secured by firebrick disks and refractive wool at both extremes, leaving most of the tube surface exposed. Both Swagelok assemblies were also supported from below to reduce the mechanical stress on the interface assembly. The heating element was powered by an ac variable transformer and current, as monitored with a standard ampmeter, was varied between 4 and 5 A (2 A on standby). A smooth ignition in the THG interface was obtained by using the following sequence: (a) The thermospray oxygen (OT) flow rate was adjusted to 500 mL/min. (b) Heating element current was increased to 6 A. (c) After 1-2 min (thermospray tube outer skin temperature 900-lo00 "C) the capillary transfer line was introduced midway in to the heated portion of thermospray tube. (d) HPLC pump flow rate (100% methanol) was rapidly adjusted to 0.5 mL/min [CAUTION: at lower heating element temperature (or insufficient heating of the thermospray tube), an accumulation of methanol in the interface due to an unsuccessful
thermospray ignition caused noisy explosionsin the optical tube; however, the interface remained intact]. (e) The hydrogen flow rate was then adjusted to 1 L/min. (f) Excess hydrogen was ignited (with a piezoelectric lighter) at both ends of the optical tube. (9) The analytical oxygen (OA) flow rate was adjusted to 200 mL/min or more until ignition of the analytical flame occurred. The hydrogen flow rate was subsequentlydecreased until extinction of the flames at both ends of the optical tube (the analytical flame should remain ignited). The hydrogen flow rate was then readjusted to its original value. The capillary depth, HPLC eluent flow rate (FR),and OT flow rate were then adjusted to obtain a stable thermospray effect (homogeneousflame accompanied by a characteristic"spray" sound). Hydrogen and OA flow rates were adjusted to optimal values. Instrument shut-down was performed smoothly by following this procedure in reverse sequence. It is helpful to retract the capillary from the heated thermospray region after the flow of mobile phase has been stopped. The use of a full-face shield is recommended until the operator is familiar with the system. Instruments. The instrumentation used for this study comprised an HPLC pump (Beckman Model 100 A), an autosampler (LKB, Model 2157), and an atomic absorption spectrometer (set at 193.7 nm; Phillips, PU9100), which was equipped with a high-energy arsenic HC lamp (Photron super lamps system, Australia) and a deterium background correction system. The optimzation experimentswere performed by removing the HPLC column and transferring the analytes from the injector to the interface via a 20 cm section of stainless steel tubing (0.04 cm i.d.). Deuterium background correction was used for theae experiments. Because background correction almost tripled the electronic background signal of the AAS detector, the chromatographic calibrations were performed without deuterium background correction. Narrow-bore stainless-steel tubing (0.01 cm i.d.) was used postinjector. Optimization. The five variables to be optimized for arsonium analytes were the flow rate of oxygen [(OT) 500-800 mL/min] and hydrogen [(H2)1.OC-2.40 L/min] to the combustion chamber, analytical oxygen flow rate [(OA) 100-240mL/min], HPLC eluent flow rate [(FR) 0.30-1.00 mL/min] and the percent (040% (v/v)) of diethyl ether (PE) or water (PW) in the methanolic mobile phase which contained also 1% (v/v) glacial acetic acid and 0.05% (v/v) triethylamine. The effects and interactions of these variables were modeled by using a half-replicate 25 composite factorial design (1416).Integrations of the atomic absorption signal for tetramethylarsonium iodide (1pg) were recorded in triplicate for each of the 27 experimental points. The results were modeled by usingthe RSREG procedure (least-squaresmultiple regreasion) of the SAS statistical software (SAS institute, Cary, NJ). The optimum operatingparameters were determined visually by using the predicted response surface plots. Interference. The AAS responses to equimolar amounts (0.4 nmol) of different arsenic-containing standards (arsenobetaine iodide, arsenocholine iodide, tetramethylarsonium iodide, dimethylarsinic acid, and arsenic pentoxide) were recorded. The possible interference of other organometallic cations (trimethylselenonium iodide, trimethylsulfonium iodide, and trimethyllead chloride) was evaluated by coinjection at 1-and 10-fold excess concentration of interferent relative to the analyte (0.4 nmol of (CH3)dAsI). Calibration. The calibration models for the three arsonium analyteswere obtained by analyzing (1WpL injections) sequential dilutions of a fresh standard solution. The limits of detection (LOD) were determined from the calibration curves (linear regressions) by using a first-order error propagation model with base-line noise normally distributed (17). Reagents and Standards. All solvents used were "Distilled in Glass" grade or "HPLC" grade (Caledon, Inc., Georgetown,ON). Certified ACS reagent grade hydrochloric and acetic acid were used. Triethylamine was purified gold label grade (Aldrich Chemicals Co., Milwaukee, WI). Other chemicals were reagent grade or better (Aldrich Chemicals Co., Milwaukee, WI). Water was double-distilled and deionized. The synthesis, purification, and characterization of tetramethylarsonium iodide (TMAs), arsenobetaine iodide (AsBet),and arsenocholiie iodide (AsChol) standards have been described elsewhere (18). Standard purity was assessed spectroscopically (MS, 'H and 13CNMR). Stock
ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990
solutions (TMAs 1.08 X lo-' g/mL; AsBet 1.00 X lo-' g/mL; AsChol 1.12 X lo-' g/mL) of these standards were prepared in methanol and kept at -40 O C as a precaution against degradation. Dilution of these standads in methanol containing 1% (v/v) acetic acid and 0.05% (v/v) triethylamine provided working standards. HPLC Conditions. The arsonium standards were separated on a cyanopropyl bonded phase (5 pm silica support, 0.46 mm i.d. X 15 cm, LC-CN, Supelco, Inc., Bellefonte, PA) using a methanolic mobile phase containing 30% (v/v) diethyl ether, 0.05% (v/v) triethylamine, and 1% (v/v) acetic acid. The injection volume in all cases was 100 pL. A number of other chromatographic approaches (ion pairing, reverse phase, cation exchange)were evaluated. Ion pairing agents tested included methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid (Aldrich Chemicals, Milwaukee, WI), and ammonium tetraphenylborate, which was synthesized by precipitation of aqueous sodium tetraphenylborate with ammonium acetate. The crude product was purified by reprecipitation from methanol with water. Stationary phases tested included Bondapak C18(0.21 cm i.d. X 30 cm, 10 pm particle size, Waters Chromatography Div., Milford, MA), Nucleosil C18 (0.46 cm i.d. X 15 cm, 5 pm particle size, CSC, Ltd., Montreal, PQ),and a weak cation exchanger (ICW, 0.32 cm i.d. x 5 cm, BDH, Inc., Montreal, PQ). RESULTS AND DISCUSSION Post-HPLC Hydride Generation. The prototype thermochemical hydride generation HPLC-AAS interface was developed as a relatively inexpensive analytical tool for studying the fate of potential biogenic arsonium compounds. Due to high spectral interference of the methanol/oxygen flame a t low AAS wavelengths, a direct thermospray-microatomizer interface previously designed for the analysis of low nanogram amounts of alkylleads (19) was inefficient for As determination. Deuterium lamp background correction was ineffective in this case. On-line hydride generation with NaBH, (13) was not investigated since the arsonium compounds of interest do not react readily with reducing agents. The thermochemical hydride generation (THG) interface presented in Figure 1represented a direct coupling of three techniques: (a) thermospray nebulization followed by pyrolysis and atomization of the organometallic analyte (19); (b) a postulated gas-phase thermochemical hydride generation promoted by hydrogen atoms, and (c) cool diffusion flame atomization (11,12). The diffusion flame atomization process was highly compatible with the THG reaction since high hydrogen flow rates were required in both processes. The atomizationof the hydride has been attributed to reaction with hydrogen radicals, which are generated in the diffusion flame. This reaction zone is formed in a spatially limited cloud of free radicals that does not extend to the AAS optical beam, virtually eliminating spectral background noise (12). To our knowledge, this is the first report of the application of a thermochemical hydride generation process to an analytical technique. The thermochemical hydride generation mechanism of the interface was supported indirectly by three observations: (a) no AAS signal was observed in the absence of the analytical diffusion flame, indicating that the species emerging from the THG was probably molecular (and volatile); (b) no signal was observed if the postthermospray hydrogen was replaced by helium; (c) inorganic arsenic(V) was also derivatized. Although the detection of the alkylarsonium compounds may result from the formation of volatile alkylarsine (partial dealkylation), only a thermochemical derivatization to arsine (ASH,) may explain the volatilization and detection of As,05 in this interface. In this postulated thermochemical process, the final product (ASH,) is rapidly stabilized by the cooling effect of the hydrogen steam; arsine decomposes at temperatures above 300 "C. Replacing thermospray oxygen by nitrous oxide (producing an intense flame exceeding 1665 "C) resulted in a complete loss of AAS signal at 500 X LOD for tetramethyl-
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arsonium. One plausible explanation for this result is a rapid degradation of the hydride at higher postthermospray temperatures. Replacing thermospray O2by air decreased the AAS response by 34%. In this case a high air flow rate (>2 L/min) was necessary to maintain the thermospray flame and the lower response may have been the result of a lower residence time of the analyte in the interface. Optimization. The interface operating parameters were optimized by using a multivariate approach based on a half-replicate 25 composite design (15,16). These experiments were carried out by recording the response of (CH3),AsI under different flow rates of thermospray oxygen (OT), hydrogen (H2), analytical oxygen, (OA), and HPLC mobile phase (FR). A fifth variable was introduced in the model to mimic typical normal- and reverse-phase HPLC eluents [0-40% diethyl ether (percent ether, PE), or 0-40% water (percent water, PW) in a methanolic mobile phase]. Although the average variation between observed and predicted responses were relatively low [8.6% and 3.3% for the normal-phase (NP) and reverse-phase (RP) models, respectively], lack-of-fit tests were statistically significant (p < 0.05) for the two models. Linear regressions on predicted vs observed data from each models were well correlated ( r = 0.8698 and 0.9814 for the NP and RP models, respectively). The accuracy of the models was considered sufficient to estimate the effect of individual variables and to determine optimum parameters visually using surface response plots. The effects of the five variables [flow rates of thermospray oxygen (OT), analytical oxygen (OA), hydrogen (HZ), mobile phase (FR), and the proportion of eluent modifier (PE) for ether or PW for water] were characterized by plotting two selected variables vs response while keeping the three other variables constant at the center of the design. The surface response plot (normal-phase model) of mobile phase (FR) vs thermospray oxygen (OT) flow rates (with PE at 20%, H2 at 1.7 L/min, and OA at 170 mL/min) is presented in Figure 2A. The response was maximum at intermediate values (OT = 650 mL/min; FR = 0.62 mL/min). The minimum response occurred at low OT and high FR. The latter conditions resulted in a fuel-rich thermospray flame and was accompanied by a slow accumulation of carbon deposits in the THG combustion chamber. A similar trend was predicted when the proportion of ether (PE) was plotted vs OT (Figure 2B). In this case, the THGAAS response was decreased by about 37% (relative to maximum) at high PE and low OT (fuel rich), due to an incomplete combustion of the mobile phase. When combined with a proper level of OT, the presence of diethyl ether in the eluent appeared to be beneficial. Thus, in the normal-phase model, the optimum response occurred at intermediate OT, FR, and P E values. The presence of water in the mobile phase (RP model) was generally detrimental (approximately 30% decrease in response relative to that observed with a similar proportion of ether). The combined effect of OT and FR (with 20% water in the eluent) is presented in Figure 2C. The minimum response was predicted (and observed) at low FR and high OT. The latter condition resulted in a relatively cool thermospray flame (fuel deficient). Decreasing OT or augmenting FR resulted in a hotter kinetic flame and an appreciable increase in response. A maximum response occurred at low OT and high FR. The combined effect of water content (PW) and OT reflected this previous observation (Figure 2D). For the arsonium analyte, a maximum response was obtained at higher flame temperatures (low water content). The temperature of this flame may affect the thermochemical hydride generation in any of at least three steps (pyrolysis of analyte, formation of the hydride, postthermospray thermal decomposition of the hydride). Without additional data, one can
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990
A
C
B
D
Flgure 2. Surface response plots of (A, C ) thermospray oxygen flow rate (OT) vs HPLC mobile phase flow rate (FR) and (B, D) percent of eluent modifier [ether (PE) or water (PW)] vs OT. The other variables were maintained constant at the center of the experimental design.
Table I. Relative AAS Responses as Compoundsa analyte
re1 response, 70
tetramethylarsonium iodide arsenobetaine iodide arsenocholine iodide dimethylarsinic acid arsenic pentoxide
100 f 1.2b 98 f 1.7 103 f 1.4 106 f 0.8 75 f 2.4
’Response to equal amounts (0.4 nmol) of the analytes recorded under optimum conditions (column removed). bStandard deviation based on three replicate analyses. only speculate on explanations for these observations. Optimum THG parameters for analysis of the arsonium analytes were estimated from surface response plots of OT vs H2 at different levels of analytical oxygen (OA) with the two other parameters [mobile phase flow rate (FR) and percent diethyl ether (PE)] kept constant a t optimum chromatographic values (FR = 0.65 mL/min; P E = 30%). These plots are presented in Figure 3. Generally, at low OT, the response was lower with a maximum occurring at more than 1.7 L/min H2. At high OT and low H2, this flame was more vigorous, but lower responses were predicted, most probably reflecting a rapid consumption of H, by excess oxygen. Increasing the flow rate of hydrogen resulted in higher responses. Estimated optimum operating parameters for the determination of arsonium compounds were as follows: OT, 700 mL/min; H2, 2.05 L/min; OA, 205 mL/min; FR, 0.65 mL/ min; PE, 30% (v/v). All subsequent analyses were performed under these conditions. Relative responses, observed for five representative organic and inorganic compounds of As (0.4 nmol), are presented in Table I. The interface provided similar responses for the three arsonium analytes [As(-III)] and dimethylarsinic acid [As(III)]. The efficiency (75%, relative to TMAs) of the thermochemical hydride generation of the pentavalent arsenic oxide analyte to arsine was essentially similar to the efficiency
Table 11. Effect of Potential Interferents on Response of (CHI),AsI’ anal*
(CH3)M
interferent (CH3),SeI (CH.&SI (CH,),PbCl
re1 amt, mol/mol
response, % loo f 1.8b
1
88 f 1.3 52 f 1.3 97 f 4.0 98 f 2.5 97 i 1.3
10
96 f 9.0
1
10 1
10
‘Response of the analyte (0.4 nmol) coinjected with equimolar or 10-fold molar excess of interferent recorded under optimum conditions (column removed). *Standard deviation based on three replicate analyses. of the wet chemical derivatization of As(V) compounds to arsine (11). The relative AAS response of tetramethylarsonium iodide (0.4 nmol) coinjected with an equimolar or with a 10-fold excess of a potential interferent, selenium (as trimethylselenonium iodide), sulfur (as trimethylsulfonium iodide), and lead (as trimethyllead chloride), are presented in Table 11. The arsenic signal was decreased appreciably (12% and 48%, respectively) by the presence of a 1- and 10-fold molar excess of trimethylselenonium. Since even the most selective extraction procedure would be likely to coextract arsonium and selenonium compounds, it will be necessary to verify the relative proportion of these compounds in the final extract and (in the remote probability of chromatographic co-elution) correct for this interference. HPLC of Arsonium Compounds. The main criteria to be fulfilled by the HPLC portion of the system were (a) a base-line separation of the analytes as well-resolved symmetrical peaks, (b) a methanol rich (>60% (v/v)) mobile phase that should be free of alkaline-earth metals (19),(c) a chromatographic performance remaining unaffected by reasonable amounts of other “onium” species which may be coextracted
ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990
B
(X lO000)
C (X l00000I
Figure 3. Surface response plots (normal-phase model) of hydrogen flow rate (H2) vs OT at different levels of analytical oxygen (OA): A, OA = 100 ml/min; B, OA = 170 mL/min; C, OA = 240 mL/min. Both FR and PE were maintained constant at 0.65 mL/min and 30%, respectively.
during the isolation of the analytes, and (d) isocratic elution,
to increase precision and facilitate automation of the method. Arsenobetaine and arsenocholine have been separated by ion-pair and reverse-phase HPLC (7).Although this particular approach was not considered compatible with our criteria (mobile phase containing sodium dodecyl sulfate; double peak for arsenobetaine), different ion-pairing approaches were investigated for separating the three test arsonium compounds. A microbore Bondapak CI8 (2.1 mm X 30 cm) column (flow rate 0.2-0.5 mL/min) was used for this purpose with different combinations of methanol (0-100%), water (0-100%), and methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, and ammonium tetraphenylborate (100-2000 pg/mL with methanol proportion >70%). The methanolic content of the chromatographiceffluent was maintained between 80% and 90% by postcolumn methanolic enrichment using a Tunion. Although very soluble in methanol/water mixtures, the analytes remained immobilized on the column in the absence of pairing agents, indicating a strong adsorption affinity for the stationary phase. The separation of arsenobetaine and arsenocholine was readily achieved (in less than 10 min) by using a high water content (SO-SO%) and moderate pairing ion concentrations (500-1000 pg/mL). However, tetramethylarsonium and arsenocholine were invariably coeluted, even using concentration gradients of water and ionpairing agents. Arsenobetaine (AsBet) and arsenocholine (AsChol) have also been separated on a Bondapak CIS column using a methanolic mobile phase (10% water) acidified at pH 3.5 with
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acetic acid (6). These conditions were highly effective for the separation of AsBet and AsChol, but coelution of AsChol and tetramethylarsonium (TMAs) remained a problem. In this case the analytes appeared to be separated mainly by a silanophilic mechanism (ion exchange interactions of analyte with underivatized silanol groups on the surface of the stationary-phase particles). The capacity factors of AsBet and AsChol were highly dependent on the acidity of the mobile phase. The addition (500-1000 pg/mL) to the mobile phase of different alkylamines (triethylamine, ethylenediamine), mixed together with different amounts of acetic acid (5-15%) and water (10-40%) in methanol resulted in the coelution of the three analytes with the solvent front. Under conditions of lower masking agent concentrations or gradients (0-30 pg/ mL), a complete separation of AsChol from TMAs was possible but the resulting chromatographicpeaks were $road and asymmetric. A similar chromatographic profile was observed when using a weak cation exchange resin based column (Polyspher ICW). Strong cation exchange chromatography (sulfonate based support) appeared to be the method of choice for separating AsBet, AsChol, and TMAs (4). However, this approach was incompatible with the THG interface because the high proportion of water in the mobile phase disrupted the thermospray effect. The relatively high proportion of organic buffer used in this technique (typically 0.1 M formate-pyridine) would also be difficult to pyrolyze efficiently. In principle, postcolumn methanol enrichment could have been used in this case. Since the maximum capacity of the THG interface was estimated at 1.5 mL/min, this approach would have necessitated reducing the flow rate of the chromatographic eluent to 0.6 mL/min or less (to allow 0.8 mL/min methanol enrichment), resulting in an appreciable loss in resolution and a longer analysis time. With optimum flow rates of 0.2-0.5 mL/min, a microbore (0.21 cm i.d.) strong cation exchange column would have been desirable in this particular case. However, this packing was not commercially available in this format. The defined chromatographic criteria appeared to be met by using a normal-phase HPLC approach, a cyanopropyl stationary phase eluted with a methanolic mobile phase (methanol-diethyl ether, 7/3) containing a silanol masking agent [0.05% (v/v) triethylamine + 1% (v/v) acetic acid]. Chromatograms of the three arsonium analytes recorded under optimum chromatographicand THG conditionsare presented in Figure 4. The addition of 30% (v/v) ether was necessary to obtain a base-line separation of AsChol and TMAs. A solvent front (s, Figure 4) resulted from the fact the solution of standards did not contain ether, resulting in a slight disruption of the thermospray effect. The addition of up to a 500-fold molar excess of ammonium acetate did not affect the retention times of the analytes or the background noise of the instrument. Because the background noise of the interface was negligible compared to that produced by the background corrector electronics, aJl chromatograms were recorded without background correction. There are a t least two reasons why background correction was not essential for this application: (a) in contrast with other flame atomizers,the diffusion flame atomizer is highly selective toward volatile hydrides; (b) the diffusion analytical flame does not reach the optical beam, resulting in a negligible spectral interference (11, 12). L i n e a r i t y , R e p r o d u c i b i l i t y , and L i m i t s of D e t e c t i o n . The limits of detection for each analyte were calculated from their calibration curves (first-order error propagation model; base-line noise normally distributed) under optimum conditions, as described previously (16,17).The linear models were highly correlated (0.9989 < p < 0.9997) in the concentration range studied (50 ng to 1 pg, as the iodide salts). The calculated limits of detection of each analyte (as the iodide salts)
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Table 111. Reproducibility of the HPLC-THGAAS at Varying Analyte Levelsa
-
0
3
6
9
12 [min]
B a
b
c
-
0
3
6
9
12 [min]
Flgrv. 4. I-@LC-THOAAS chromatograms of (a) arsenobetaine (b) arsenocholne, and (c) tetramethylarsonium (A, 100 ng of each; B, 1 pg of each). These chromatograms were recorded under optimum conditions [OT, 700 mL/mln; H2, 2.05 L/min; OA, 205 mL/min; FR, 0.65 mllmln; PE, 30 % (vlv)]. A sokent front signel (8) resulted from the fact that the solution of standards did not contain 30% ether.
were as follows: AsBet = 22.7 ng; AsChol = 26.7 ng; TMAs = 14.8 ng. This LOD may be expressed in terms of the free cations (which is more appropriate to real samples); (CHd&+CH,COOH = 13.3 ng; (CHd&+CH2CH20H = 14.5 ng; (CH&As+ = 7.6 ng. These LODs were similar to that observed by using an HPLC-ICP system (20). The short-term reproducibility of the THG interface (basedon three replicate analyses) for different concentrations of each analyte is reported in Table 111. The long-term reproducibility (6 h; n = 6) recorded a t 10 X LOD was as follows: AsBet, 6.0%; AsChol, 2.3%; TMAs, 6.5%. The limits of detection provided by this automated HPLC-THGAAS were considered sufficient for routine analysis of arsonium metabolites in marine organisms in which they may occur at relatively high concentrations (typically 0.1-16 mg/kg).
amt, ng
AsBetaine
% RSDb AsCholine
TMeAs
50 100 500 1000
12.9 1.5 1.4 4.2
14.6 2.2 2.6 2.0
13.5 5.0 6.2 1.0
a Recorded under optimum conditions. bRSD,relative standard deviation based on three replicate analyses.
CONCLUSION Since the cool diffusion flame atomizer has been shown to provide subnanogram sensitivities for As and Se at total gas flow rates exceeding 3 L/min (11,12),it may be aasumed that the performance of this interface is limited by the efficiency of the thermochemical hydride generation and hydride transport processes. As suggested by the strong quenching of this process in a nitrous oxide supported flame, the performance of the THG interface appears to be susceptible to excessive postthermospray temperatures. Thus, it is reasonable to predict that further research on this aspect may result in subnanogram limits of detection for hydride-forming elements. Different chemical and thermochemical approaches are under study to characterize this thermochemical hydride generation process and determine other applications for this novel approach to HPLC-AAS interfacing. LITERATURE CITED Kurosawa, S.; Yasude, K.; Taguchi, M.; Yamakasi, S.; Tcda, S.; Morlta, M.; uehiro, T.; Fuwa, K. A&. Bkl. Chem. 1980, 44, 1993. Cannon, J. R.; Edmonds, S. J.; Francesnoni, K. A.; Raston, C. L.; Saunders, J. B.; Skelton, 8. W.; White, A. H. Aust. J . Chem. 1981, 34, 787. Shiomi. K.; Shinagawa, A.; Igarashi, T.; Yamanaka, K.; Kikushl, T. Experient& 1984, 40, 1247. SMOml, .K.; Kakehashi, Y.; Yamanaka, H.; Kikushi, T. Appl. Orgemmet. chem. 1987, 1 , 177. Francesconi, K. A.; Micks, P.; Stockton, R. A,; Irgdlc, K. J. chemosphere 1985, 14, 1443. Lawrence, J. F.; Mlchaik, P.; Tam G.; Conacher H. B. S. J . Agdc. Food chem.1988, 34, 315. Beauchemin, D.; Bednas. M. E.; Berman, S. S.; Mclaren, J. W.; Siu, K. W. M.; Sturgeon, R. E. Anal. Chem. 1988, 60, 2209. Low, G. K. C.; Batley, G. E.; Buchanan, S. J. J . chrometog. 1988, 388, 423. Stockton, R. A.; Irgoiic, K. J. Int. J . Envkon. Anal. Chem. 1979. 6 , 313. Welz, B.; Melcher, M. Anakst 1983, 708, 213. Slemer, D. D.; Koteel, P.; Jawlwala, V. Anal. Chem. 1978, 48, 836. Dedina, J.; Rubeska, I.specb;ochkn. Acta 1980. 358, 119. Tye, C. T.; Hasweli, S. J.; ONeH, P.; Bancroft, K. C. C. Ana/. CMm. Acta 1985. 169, 195. Forsyth, D. F.; Marshall. W. D. Ana/. Chem. 1985, 57, 1299. HUI. W. J.; Hunter, W. G. TechnomebJcs 1968, 8, 571. Blais, J. S.; Marshall, W. D. J . Anal. At. spectrom. 1989, 4 . 641. Foley, J. P.; Dorsey, J. G. Wwomarographla 1984, 18, 503. Momplaislr, G. M. M.Sc. Thesis, McOill University, Montreal, 1990. Blais. J. S.; Marshall, W. D. J . Anal. At. Spectrom. 1989, 4 , 271. Irgolic, K. J.; Stockton, R. A.; Chakraborti D. SpectrocMm.Acta 1983, 388, 437.
RECEIVED for review February 5,1990. Accepted February 12,1990. Financial support was from the Natural Science and Engineering Research Council of Canada (STRGP036).
