Methods Based on Elemental-Specific Detection

Investigations using the hyphenation of GC [38] or HPLC [39] to ESD were first carried out in the late 1970s and early 1980s. Refinement of the approach has taken place since then and other separation methods, such as CE and SFC have been developed. Early reviews of different separation approaches coupled to ESD or MS included the use of GC [40], HPLC [41], and SFC [42]. Element-specific detectors such as ICP-MS or techniques based on AAS or AFS are used because of their analyte specificity, provision of quantitative data using elemental standards and potential to provide suitable limits of detection (LODs) for environmental and biological sam- ples. In practice, AAS is generally not sensitive enough without VG to be used for real samples and AFS, whilst offering suitable LODs for speciation studies [43], is limited to elements forming stable hydrides or elemental species. ICP-MS provides the most versatile detection system because it can be coupled to numerous different chromatography techniques, delivers suitable LODs, offers a long linear calibration range (although this may be limited by the separation technique), is tolerant to complex matrices, offers multi-elemental and isotopic analysis and provides quantitation based on elemental standards.

Common problems involving ESD include: identification of unknowns through a lack of standards; unrecognized coelution of different species containing the same metal(loid); and retention times affected by sample matrices. One of the first major issues that became apparent was the diffi- culty in identification of unknown species and the inherent possibility of misidentification. This is one of the main drivers for the development of complementary methods based on molecular MS. Identification using ESDs relies on the availability of authentic molecular standards of high purity which are used as retention time markers. However, even when these are available it is possible to make wrong assignments, particularly if the spiking procedure is not carried out with care. A good example of this relates to the misidentification of organotin compounds in the marine environment [44]. In this case a number of techniques based on sample derivatization followed by GC separation (GC-QF-AAS, GC-FPD, GC-AES, and GC-MS) were used to identify the compound responsible for a peak eluting between the derivatives of monobutyltin (MBT) and dibutyltin (DBT). It had initially been proposed that the peak was due to the presence of a mixed methyl- butyltin compound, which would have indicated that an important trans- formation pathway was operating in the biogeochemistry of OTCs. How- ever, after a concerted analytical programme involving a number of laboratories it was found that the unidentified compound was actually due to monophenyltin, probably resulting from the degradation of triphenyltin (TPhT), a widely used pesticide.

The most important requirements for interfacing the separation system to the ESD are that the analyte is quantitatively transferred from one to the other without loss or rearrangement. Figure 1 shows a schematic diagram of the on-line coupling of HPLC or GC to ICP-MS. Conventional ICP-MS operates on liquid samples that are introduced via nebulization at a flow rate of 0.1 to 1 mL min 1. With liquid-based separations using HPLC, a suitable length of tubing can be used to couple the column to the nebulizer. Alter- natively, for some elemental species HPLC can be hyphenated to ESD via VG (see Section 3.5). With the other separation systems (GC, CE, SFC) the interface has required development work to be carried out to accommodate the differences between the separation system and the requirements of the ICP-MS.

The main difficulties when coupling HPLC to ICP-MS involve eluents containing a high proportion of an organic modifier, because this can destabilize the plasma, necessitating a cooled spraychamber (5 to 15 1C) or low flow conditions, to reduce the solvent load. Oxygen addition is required to eliminate the deposition of carbon on the sampling cone and maintain the transmission of ions through the cone orifice. To withstand the extra wear generated, a platinum tipped sample cone has to be used. The advent of low-flow and desolvating nebulizers has helped with coupling HPLC to ICP-MS and more recent applications have not used cooled spray chambers. This type of sample introduction system allows the use of gradient elution, which makes possible shorter chromatographic runs and more ver- satile separation systems. Recent developmental work has produced a sheathless interface using a microflow total consumption nebulizer, which facilitates the use of eluents containing 100% organic solvent, without spray chamber cooling or oxygen addition [45]. This makes the coupling of

Inter -face Mass Analyser Detector Quadrupole Computer -Control -Acquisition -Analysis HPLC System Cooled Spray Chamber Nebuliser GC System PLASMA Heated transfer line PEEK tubing

Figure 1. Schematic diagram of the coupling used for the hyphenation of GC or HPLC to ICP MS.

low-flow capillary HPLC separations to ICP-MS possible and offers sig- nificant advantages over conventional columns because small sample volumes (nL) can be used, the chromatographic system provides enhanced peak resolution with a better signal-to-noise ratio and consequently a lower LOD. Coupling GC to ICP-MS requires heating of the transfer line to a tem- perature higher than that used in the separation so as to prevent cold spots, which lead to peak broadening or complete retention of the analyte within the system. The first use of a heated transfer line was described in 1992 [46,47] and consisted of an aluminum bar with a slit, in which the capillary column was contained, before introduction into the central channel of the torch. The necessary argon make-up flow was heated in the GC oven prior to its introduction through a T-piece and sheathed the column, helping to avoid condensation in the transfer line. This interface was successfully applied to the analysis of high boiling point compounds such as Fe, Ni, and V containing porphyrins [48,49]. Another interface design in which a heated quartz transfer line was inserted through the torch to the base of the plasma has been developed commercially [50]. Recently the construction and eva- luation of a low cost interface which could be adapted for use with most GC and ICP-MS instrumentation has been described [51].

The main advantage provided by using GC separations is that around 100% of the injected sample reaches the detector and because no liquid is introduced the plasma is not cooled. With HPLC only a few percent of the sample reaches the plasma due to the inefficiency of conventional nebulizer- spraychamber configurations and the wet aerosol cools the plasma, reducing the energy available to ionize the analyte. In general GC methods have better S/N ratio characteristics than HPLC methods, because of the sharp and narrow peak shapes generated. Another important characteristic of GC- ICP-MS is the ability to perform multi-elemental speciation studies, which is generally not possible with HPLC because of the limitations in chromato- graphic selectivity.

With GC separations the volatility of the analyte is the principle factor determining how long the analyte stays on the column, so as long as the chemical species are stable and volatile they can be separated regardless of the element. With HPLC separations other properties such as polarity determine how the chemical species behave, making it difficult to develop separations that accommodate the diverse range of OMC properties. Capillary GC separations also have the potential to deliver better compound resolution compared to HPLC. The main difference between the two approaches is that GC requires an extra step, so that the generally ionic, low volatility compounds are converted to a stable volatile form, with HPLC the target analytes are determined directly. The consequence of this extra deri- vatization step is that there is a significant chance the analyte could be lost or an artefact formed during the reaction.

Derivatization reactions, especially aqueous ethylation with sodium tet- raethyl borate (STEB), used when GC separation is employed prior to detection of Hg compounds have been implicated in the formation of arte- facts [52]. This derivatization step is inhibited by high concentrations of chloride ions [24]. The high stability of the MeHg chloro complex which is formed in high chloride-containing samples has been suggested as an explanation. The ability of halide ions to interfere with the ethylation reaction is of particular importance when MeHg extraction using HCl is employed and not just when seawater samples or other high chloride con- taining samples are analyzed [53]. Chloride and bromide ions have been reported to convert MeHg into Hg(0) and iodide promotes a dis- proportionation reaction of MeHg to produce both Hg(0) and Hg(II) [52]. The same study showed that derivatization using propylation did not cause this conversion.

The main advantage of HPLC compared to GC is that there is no need to derivatize the compounds prior to analysis. However, acidic or alkaline sample extracts do need pH adjustment when a silica-based column is used, otherwise the chromatographic medium could be damaged. This pH adjustment has been implicated in the artefactual formation of MeHg from Hg(II) [8]. Mercury compounds are notorious for exhibiting memory effects, i.e., adhering to internal components of HPLC instrumentation and various mobile phase additives have been used to try to reduce this. One very effective method to eliminate poor peak shapes, high blank values and non-eluting compounds, is to use polyetheretherketone (PEEK) instead of stainless steel components in the HPLC system and include 2-mercaptoethanol (2-ME) in the eluent [54]. Another sulfur-containing reagent used to reduce these effects is cysteine [25]. Other problems related to the analysis of Hg in biological and environmental samples have been encountered and these have been reviewed [55]. Figure 2a (see Section 3.3) shows a typical chromatogram obtained for the analysis of Hg species by using HPLC-ICP-MS when using 2-mercaptoethanol to reduce peak tailing.

SFC uses a liquefied gas as the eluent and programmed changes in pres- sure to facilitate separation, in a similar way to temperature programming in GC separations. Supercritical fluids have critical temperatures (temperature above which the fluid cannot be liquefied) below 200 1C and densities of the order 0.1–1 g L 1at pressures of 1000–6000 psi. Carbon dioxide is the most common eluent for SFC analysis of metal(loid) species and in some appli- cations has been doped with methanol. SFC-ICP-MS overcomes some of the limitations of HPLC and GC because it can be used to rapidly separate thermally labile, non-volatile, high molecular weight compounds and affords lower LODs. The interface between SFC and ICP-MS is commercially available and involves a restrictor to maintain the high pressure required for

the separation system. However, only a few applications have used SFC- based methods and the majority of these have focused on the determination of OTCs in marine samples [56,57].

CE is a family of related techniques that employ narrow bore (20-200 mm in diameter) capillaries to perform high efficiency separations [58], facilitated by the application of a high voltage to the capillary, which generates elec- troosmotic and electrophoretic flow. The technique has been coupled to ICP-MS and ESI-MS [59] for the measurement of OMCs in biological and environmental samples. The initial difficulties in designing a suitable inter- face to couple CE separations with ICP-MS were centered on the high flow- rate requirements of conventional ICP nebulizers and the low-flow rate nature of CE. The suction generated with the conventional self-aspirating nebulizers, caused a loss in chromatographic resolution and the necessity to maintain an effective electrical connection to the end of the capillary posed problems. These difficulties were overcome by using a low-flow nebulizer and a small make-up buffer flow with an earth connection [60]. The main advantages of CE for speciation analysis include: minimal species interaction with separation media due to its absence from the capillary; potential to measure neutral, variably charged, and organometallic species in a single run; low sample consumption; and a high separation efficiency compared to other liquid chromatographic methods. However, because of the small sample size used it is difficult to detect the species present in real samples unless a low LOD detector is available.

3.3.

Methods Based on Molecular Mass Spectrometry

Molecular mass spectrometry has been used in conjunction with some of the above mentioned chromatographic techniques for the analysis of OMCs. The most commonly used ionization techniques for HPLC and CE are atmospheric pressure ionization (API), of which there are two main variants, electrospray ionization (ESI) and chemical ionization (APCI). Traditional mass spectrometry using electron impact (EI) ion sources have been used with GC separations. The main characteristics of these molecular detection methods when used for the analysis of OMCs include: ionization specific to the analyte molecule; possibility for structural studies via tandem MS ana- lysis; potential for high mass accuracy characterization; availability of a wide range of commercially available hyphenated instrumentation; wide m/z range analysis; and low LODs, although not as low as for ICP-MS.

The advantage of molecular detection is that it is possible to identify unknown chemical species in situations where standards may not be available and it offers the potential for structural elucidation. When using

API-MS for the analysis of environmental or biological samples it can suffer from significant matrix effects, so may require extensive sample clean-up procedures to be used, to eliminate the effect and reduce the formation of sodiated and potassiated ions. Matrix effects are still a difficult problem to contend with in API-MS analysis, where a ‘‘soft-ionization’’ process is used for ion generation. Unlike API-MS, ICP-MS is such a ‘‘hard-ionization’’ process that suppression of ion formation by the sample matrix is not considered a problem. Hence, the major shortcomings of ESI-MS compared to ICP-MS are the much poorer LOD and the adverse effect of the matrix present in biological and environmental samples.

The majority of methods using API-MS involve ESI-MS which was first developed in the mid-1980s [61,62] and used for the analysis of large molecular weight, non-volatile biomolecules and more recently for small polar metabolites [63]. In the case of organometallic analysis ESI was initially used for the determination of small polar or ionizable compounds such as tributyltin (TBT), or As species, but the greatest impact of ESI-MS has been made in the analysis of much larger molecules, particularly metalloproteins. The use of ESI-MS for the analysis of OMCs has been reviewed [64,65]. The complementary ionization source to ESI is APCI and this has found some limited use for the analysis of OMCs; Figure 2b shows the detection of mercury species by APCI-MS, after HPLC separation using 2-ME in the eluent and Figure 2c the APCI mass spectrum for the MeHg peak, corresponding to an adduct between MeHg and 2-ME and clearly shows the isotopic pattern for Hg.

The most important technical difference between ICP-MS and modern API instrumentation is the possibility to carry out tandem API-MS/MS experiments. The ions formed in the source are sampled in to the first quadrupole and then either the molecular ion or a fragment ion is isolated in a collision cell containing an inert gas with a collision voltage applied. Depending on the ion and the voltage the sampled ion is further broken down into different fragments. This approach, termed collision-induced dissociation (CID), results in highly specific analysis, provides the lowest LODs and the ability to investigate the structure of the molecule of interest. This technique has made a significant impact on our understanding of the biogeochemistry of As in the marine environment, where a range of As-containing sugar compounds are found. By using tandem MS, with an ESI source it is now possible to directly characterize these novel arsenicals directly after HPLC separation [66]. Until the advent of ESI-MS/MS these marine arsenicals were investigated using a natural products approach, whereby large quantities of material are extracted to isolate sufficient of the As compound for identification by NMR [3]. Electrospray principles and general applications were reviewed extensively in 2000 [67].