Element Speciation Analysis Using Capillary Electrophoresis: Twenty

Oct 11, 2012 - After 6 years there, finally as a Senior Research Fellow, he earned a Doctor of Science degree in chemistry in 1991. In 1991, he became...
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Element Speciation Analysis Using Capillary Electrophoresis: Twenty Years of Development and Applications Andrei R. Timerbaev* Vernadsky Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, Kosygin Str. 19, 119991 Moscow, Russian Federation 7. Speciation Studies Related to Metal Complexation 8. Speciation Analysis by Chip-Based Electrophoresis 9. Summary and Future Outlook Author Information Corresponding Author Notes Biography Acknowledgments Abbreviations References

CONTENTS 1. Introduction 1.1. Current State of Speciation Measurements in Analytical Chemistry 1.2. Role of Capillary Electrophoresis in Species Separation, Characterization, and Determination 2. Common Types of Element Species 3. Separation Methodology for Species Analysis Using Capillary Electrophoresis 3.1. Approaches for Maintaining Species Integrity 3.2. Approaches for Improving Resolution 3.2.1. Species Derivatization 3.2.2. Adjustment of Electrolyte Composition 3.2.3. Modification of the Electroosmotic Flow 3.2.4. Use of Supplementary Separation Mechanism 3.2.5. Amendments for Real-Sample Analysis 4. Detection Systems 4.1. Couplings to Element-Selective Techniques 4.1.1. Inductively Coupled Plasma Mass Spectrometry 4.1.2. Inductively Coupled Plasma Atomic Emission Spectrometry 4.1.3. Atomic Fluorescence and Atomic Absorption Spectrometry 4.2. Combinations with Molecule-Selective Detection 4.3. Nonselective Detection Methods 5. Sample Preparation for Reliable Speciation Analysis 5.1. Sample Cleanup 5.2. Sample Preconcentration 5.2.1. Off-Line Preconcentration Techniques 5.2.2. In-Line Preconcentration Techniques 6. Real-World Speciation Applications

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1. INTRODUCTION

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1.1. Current State of Speciation Measurements in Analytical Chemistry 779 780

Due to a growing awareness of the importance of element speciation in a wide range of analytical applications,1 the knowledge of the distribution of defined chemical species in which an element exists in a sample is now a mandatory requirement in most analytically oriented chemical investigations. Relevant important areas include toxicology, environmental chemistry, geochemistry, clinical chemistry, etc. The reasons are 2-fold. The first is brought about by a growing need to identify the chemical nature of the relevant element species and to determine their exact quantities. The necessity for this knowledge is due to the fact that the actual chemical form (e.g., oxidation state or bonding type) and its content, rather than the total concentration, are what in fact governs the ultimate environmental and biological behavior of any given element. In many cases, one form of the metal or metalloid can be toxic, whereas the same metal(loid) when in a different form is nontoxic and even necessary for sustaining an ecosystem or the efficient functioning of a living organism. Therefore, the determination of the concentration of a specific chemical form, such as Cr(VI) instead of the total chromium content, organic mercurials rather than elemental mercury, or free versus the bioligand-bound metallodrug, is essential for the comprehensive interpretation of their ecotoxicological effects or an assessment of their physiological impact on different organisms. In other words, only an awareness of element speciation provides us with an in-depth insight into their toxicity, mobility, bioavailability, essentiality, and other critical properties. In addition, species determination helps in developing successful strategies for waste management and

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growing demand for the identification of unknown species, elemental ICP-MS is well complemented by the synergetic use of molecular MS, typically with electrospray ionization (ESI), suitably integrated in HPLC-based speciation schemes. However, it stands to reason that while satisfying most of the above-mentioned requirements, the HPLC−MS methodology has not gained the status of a routine speciation tool. Some of the reasons are because of its complicated design, the need for expensive instruments, and quality control issues. But an even more critical issue comes from the nature of HPLC itself. The possible instability of species due to interaction with the stationary phase (or column material) or incompatibility with the mobile phase in use as well as the marginal vulnerability to matrix interferences is the issue. The problem of undergoing transformations can be particularly troublesome in the case of labile complexed forms that might suffer cleavage of their metal−ligand bonds. These recognized limitations of HPLC have impelled analysts to seek alternative separation techniques suitable for hyphenation with MS or other (element-specific) detectors. One such technique that has experienced confident growth since the mid-1990s and has attained the state of complementarity in the field to be discussed herein is capillary electrophoresis (CE).

decontamination, since the removal of metal pollutants is highly species-dependent. Although less relevant in the context of this review, there is also a rising recognition of the importance of metal speciation in order to achieve a better control of many industrial processes. The second reason why analysts nowadays are increasingly requested to disclose speciation information is that it is only over the last two decades or so that the arsenal of experimental technologies and tools of analytical chemistry has been developed to acquire the separation/detection sophistication and power required to distinguish and measure individual species, often occurring at trace levels and in a complex matrix environment. In order to make species-selective measurements reliable, an analytical technique should meet a number of requirements. The most important include: (i) the ability to differentiate (multiple) ionic and nonionic element forms in often complex mixtures, i.e., a high separation (or signal resolution) power is essential; (ii) the potential to identify the species with respect to their functionality (e.g., the presence of a target metal) and structure; (iii) a high detection capacity in order to make the quantification of trace amounts of the species possible; (iv) only a minor impact of the analytical system on the original distribution of the element species in the sample, so that no loss of speciation information is encountered during analysis; this is also the reason why short analysis times are preferred; (v) a good tolerance to complex matrices, which is the case of many environmental and biological samples; otherwise, the integration of a sample preparation procedure that preserves the analytes against changes in speciation is necessary; and (vi) practicability in terms of easy implementation, low running costs, minute sample volumes (important for certain biosamples), high throughput, minimum waste requirements, etc. Many analytical methods have been tried for speciation studies. However, it soon became evident, through a series of continuing conceptual and technical developments, that only hyphenations of separation techniques and element- or molecule-selective detection systems provide the basis for trustworthy speciation analysis. Among the proven combined techniques, high-performance liquid chromatography (HPLC)in various separation modesdirectly coupled to inductively coupled plasma mass spectrometry (ICP-MS) is unquestionably a premier technique.2 HPLC−ICP-MS is in fact more suited for speciation analysis than for total element determination, and it enables most sensitive and versatile speciation measurements. In situations where species of different elements need to be determined simultaneously in one sample, the multielement detection capability of ICP-MS is clearly of merit. The use of HPLC−ICP-MS couplings can be extended for trace species concentrations by employing a highresolution (sector-field) mass spectrometer. This type of instrumentation also helps resolve the problem of spectral interferences, which render certain elements difficult to analyze using standard quadrupole ICP-MS. Alternatively, the latter instrument can be equipped with a dynamic reaction/collision cell. There is also the possibility that species quantification could be greatly improved in terms of precision and accuracy by taking advantage of isotope-dilution methods. To address a

1.2. Role of Capillary Electrophoresis in Species Separation, Characterization, and Determination

Several advantages of CE, which are commonly referred to in comparisons with HPLC and were in fact the impetus for its development in the field of speciation analysis,3 are (i) gentle, species-friendly separation conditions; (ii) relative freedom from many environmental and biological matrices that can be analyzed with zero or minimum pretreatment; (iii) greater resolving power (and thus ability to differentiate more species, ranging from small metal ions to large biomolecules with metal functionality, within a single run); (iv) exceptional possibilities for dealing with microsamples; and (v) cost efficiency. Indeed, in CE, the separation process takes place inside capillaries containing no stationary phase. Furthermore, typical capillary electrolytes are low-conductivity buffer solutions, no extreme pHs being involved, and thus often compatible with the matrix (native) surroundings of element species. The advantage of using such separation hardware and media is that it prevents the species from losing integrity in the CE system. In addition, since the risk of fouling the open-tubular capillaries is less than that for the packed columns, samples can be introduced directly (or after only a minor cleanup). As a consequence of the flat flow profile resulting from CE (unlike the parabolic flow profile in HPLC), no mass transfer between phases, and negligible latitudinal diffusion in capillaries, the analytes are separated as sharp zones. This enhances the CE separation efficiencies and hence makes the number of species separated in one run greater than that achieved in HPLC. Because typical capillary internal diameters are on the order 50−100 μm, the necessary sample volumes are as low as a few nanoliters. It should be noted that along with lower instrumental and reagent consumption costs, there is no need for relatively expensive columns, and this ensures that CE analyses are comparatively inexpensive. One inherent advantage of CE is that it involves a different separation principle than HPLC, namely, the chargeto-size differences of the analytes. This advantage is not at first obvious. Rather it presents a basis for an orthogonal speciation concept for the unambiguous identification of elemental species. It is also a somewhat controversial issue as to whether 779

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2. COMMON TYPES OF ELEMENT SPECIES Depending on their chemical nature, all relevant element species can be divided into the following major categories: (i) metal ions in different oxidation states, e.g., Fe(II) and Fe(III); (ii) metal ions versus metal−ligand complexes [here “ligand” is to be taken in a broad sense, covering inorganic, organic, and biological (macro)molecules bound to a metal via coordination bond(s)]; (iii) different complexes of one metal (with the same stipulation as above), e.g., cis-diamminedichloroplatinum(II) (known as cisplatin) and its hydroxy metabolite, or certain metalloproteins whose functions are influenced by metal composition and content; (iv) organometallic compounds (e.g., CH3Hg+, C2H5Hg+, etc., often in the presence of a free metal ion, Hg2+); (v) oxoanions and other (bio)forms of metalloids (most typically, As and Se); and (vi) nonmetals such as common anions (NO2− and NO3−) or phosphorus-containing biomolecules. Another class of species, whose separation and characterization is also accessible by CE, is that of inorganic nanoparticles. Without wishing to enter into a controversy, we consider these to lie within the scope of fractionation analysis1 and thus are less relevant to the topics covered in the present review. Rather, the interested reader is referred to recent literature treatments on the CE of nanoparticles.16 Such a great variety of analytes poses strict requirements on the entire methodology of CE analysis. For example, some species of interest might incline toward compositional or concentration alterations in the CE system (or even at the stage of sample preparation) due to their instability, lability, adsorption, etc. Other element forms, because they have similar charge-to-size ratios, are difficult to separate. Not all the species of concern possess a marked analytical property, such as strong absorbance, or isotopes sufficiently abundant (or free of spectral interferences) to make quantification attainable. Therefore, the main part of this review focuses on methodological approaches modified with a view to keeping species intact, to ensure their complete separation (even from complex matrices), and to improve the detection sensitivity.

or not CE separations are faster than those performed by HPLC. However, using a microchip4 or short-capillary format5 should definitely yield operational rewards by substantially shortening the separation time. On the other hand, it should be noted that sensitivity is one of the most serious shortcomings of CE when compared to HPLC. As an inevitable result of the very small volumes of sample injected, concentration detection limits (DL) are typically 2−3 orders of magnitude worse. In practice this may render CE unable to perform real-world speciation analyses. This sensitivity limitation can be overcome by coupling CE to ICP-MS,6 which was harmonized by the recent commercialization of interface systems. Although the method does not lend itself to hyphenation with ICP-MS as easily as does HPLC, the selection of suitable separation and interfacing conditions does allow the sensitive determination of analyte species. Additionally, the beauty of CE as a separation strategy is that it can be integrated with preconcentration techniques, operating in an inline fashion, whereby the analytes are enriched (stacked) and separated using the same capillary. An array of complementary stacking mechanisms exist that offer significant enhancements in sensitivity, regardless of the type of sample matrix. A molecular ESI-MS detector is more customarily coupled to CE than an ICP-MS system. This provides the necessary selectivity required for species identification. Yet prior to such measurements, one can still differentiate species with respect to their net charge, i.e., between anionic, cationic, and neutral forms, as based on their migration order. While not a structural characteristic, the charge state might prove important, for example, for the assessment of the metabolic pathways of various species. It should be noted that the fact that electrophoretic separation is ruled by the effective charge of analytes does not disqualify noncharged species from consideration. This is due to the fact that the separation mechanism can be easily amended (to an electrokinetic chromatography mode) by the incorporation of an appropriate electrolyte additive. The progress made in the application of CE over the 20 years since its introduction into species-selective analysis7 accentuates the necessity to summarize and critically discuss the recently accumulated data. Such a review appears very timely because since the first review literature that examined the first decade of CE research as a method for speciation analysis,3 there have been only two full overviews covering the entire field, the most recent being in 2003.8 However, several related publications dealt with the CE analysis of single-element species such as sulfur,9 selenium,10 mercury,11 platinum,12 or other metal ions13 as well as their interaction with biomolecules.14 Other reviews are available only for Chinese-speaking readers.15 Because of this, a particular emphasis is placed here on providing coverage of the latest literature, with due account of examples clarifiying the speciation scope of current CE procedures, appropriate handling of any technical issues and limitations, adequate method performance characteristics and acceptance limits, as well as the rationale used in any validation challenges. Special consideration is also given to the separation, detection, and sample preparation strategies required in order to meet “best element-speciation practice”. Finally, possible future research trends and developments in this rapidly expanding area, which, in the author’s opinion, could in fact ensure that CE attains the status of a comprehensive analytical technique, are presented.

3. SEPARATION METHODOLOGY FOR SPECIES ANALYSIS USING CAPILLARY ELECTROPHORESIS 3.1. Approaches for Maintaining Species Integrity

It may be superfluous to emphasize that the results of any speciation analysis are only trustworthy when no change in species distribution occurs during the separation process. In other words, the generic speciation in the sample is preserved until the detection step. For this reason, when selecting a separation system, the challenge is not only electrophoretic resolution of the species in question (between themselves and the matrix components, as discussed later in section 3.2) but also to ensure that they retain sufficient resistance against interconversion, i.e., changes in partitioning of an element among its species. This makes the selection of suitable separation conditions more challenging than in common CE analysis. As soon as the environment of the species during analysis differs from that in the original sample, this mismatch could shift the equilibrium existing between the (metal) species and 780

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nite38e). This is due to its oxidizing properties, and hence, species stabilization (e.g., by adding 5% v/v propanol38a or 0.1% v/v formaldehyde38e) is required. In order to prevent metal-catalyzed oxidation of sulfur-containing amino acids, 0.1 mM EDTA was added to the electrophoretic buffer.40 Even for specific analytes, such as peroxynitrite, existing at extremely high pHs, suitable electrolytes can be developed to maintain the stability.41 However, the situation turns out to be different in CE work concerned with distinguishing free metal cations. Complexation prior to electrophoresis is a common remedy for the prevention of hydrolysis and electrostatic analyte−wall interactions. In addition, this approach helps to resolve and detect metal charged states or inorganic vs organometallic forms (see below). However, as mentioned above, the metal complexes involved may be partially or completely decomposing during the separation process. To obviate this negative effect, it almost always proves satisfactory to add the precapillary derivatization agent also to the capillary electrolyte.34c,42 Alternatively, (highly) acidic electrolytes can be used to overcome the difficulties of species hydrolysis and polymerization. Some examples are the noble metals,43 various oxidation states of plutonium and neptunium,44 uranium(VI),45 or technetium(IV).46 In order to maintain accurate sample speciation of, for example, Cr(III) hydrolytic polymerization products, noncomplexing separation electrolytes, such as a LaCl3 solution,47 have proven successful. Otherwise, in order to infer structural information about the hydrolyzed Cr(III) species, they first had to be isolated by an ion exchange step.48 Adsorption onto the capillary wall is typically manifested in peak tailing, as well as a bias in the electrophoretic mobility data and in the quantification of species. Incorporation of an ionic surfactant49 or an organic solvent or the use of a concentrated (up to 100 mM)24d,36 or a low pH buffer50 may help eliminate these problems. This was particularly demonstrated by the improved peak shapes of Se(VI),51 organolead forms,34c and organotin cations52 as a result of applying an electrolyte containing, respectively, 0.5 mM sodium dodecyl sulfate (SDS), 2% v/v methanol, and 8 × 10−6 M cetyltrimethylammonium bromide (CTAB). Another important factor related to the stability of analyte species is the analysis speed. Any means that results in shorter separation times and less capillary conditioning time between runs should be tried to reduce the likelihood of analyte (bio)degradation in the sample. As a good example of this strategy to mention,53 using a short-end multiple injection mode decreased the total analysis time to less than 3 min per analysis and thus guaranteed the stability of the anionic species in saliva samples.

hence hinder the characterization of a given speciation pattern. Disequilibrium effects may be particularly troubling in CE analyses dealing with kinetically labile metal-complexed forms.17 A relevant theoretical treatment and a diagram defining the relative stability of metal−ligand complexes during CE separation can be found in ref 18. After applying a high separation voltage, the ligand leaves the sample zone, giving rise to electric-field-induced dissociation and, possibly, arriving at a new chemical equilibrium before detection. In each of these cases, clearly the resultant electropherogram does not reflect the true speciation. Fortunately, the phenomenon of disequilibrium bias is less marked for the biomolecular complexes of metals such as metalloproteins. This is largely due to the fact that a protein acting as a macroligand does not change its charge and shape significantly nor its mobility upon complexation, in contrast to low molecular mass ligands. Therefore, even though the dissociation takes place, free protein remains in the sample zone and maintains equilibrium conditions. In this context, it should be pointed out that a wide range of electrolytes are available for use with CE techniques. In most cases this allows the selection of an electrophoretic medium that can guarantee the species constancy. An obvious approach that should be mentioned in the first instance is to adjust the electrolyte composition as closely as possible to conform to the matrix of the native sample. For instance, in the speciation analysis of a variety of biological samples, the electrolyte pH is typically kept within the physiological range of around 7.4. Literature reports on a diversity of such samples: serum,19 plasma,20 and red blood cells21 or their lysate,22 cytosol of brain,23 liver24 or pancreas,24b tissues,25 plants26 and their cells,27 and others.28 Using this approach, the demetalation of metalloproteins, such as metallothioneins (MTs),23b,29 can be successfully circumvented, although not in all cases.30 Likewise, the adjustment of the electrolyte pH to around 8 when analyzing natural waters or using high-salt electrolytes, similar in composition to saline environmental waters, may be recommended in order to help minimize the probability of species transformation and interactions with uncoated capillary walls. If the information of interest is the speciation in biological or natural metal−ligand systems, any complexing electrolyte additive may in fact complicate its determination and hence must be avoided. Therefore, biologically compatible buffering agents, usually Tris19b,21,23a,b,24b,25,26b,27,28,29b,30b,31−34 or similar buffers (HEPES or N-2-hydroxyethylpiperazin-N′-2-ethanesulfonic acid,24a,b,e,f,35 Tricine,24d,31a etc.) exhibiting weak or negligible complexing properties, are given preference over, for example, phosphate-based electrolytes. This makes it easier to reduce the risk of releasing the metals from their binding forms. The complex formation between Eu(III) and humic acid was studied using acetate buffer electrolyte fortified with 100 mM acetic acid to stabilize the resulting europium complex.36 Careful purification of the electrophoretic buffer was shown to be critical in order to preserve the MTs against the exchange of the Zn with either Cd or Cu ions.25b Because of the susceptibility of these S-rich analytes to oxidation, it is necessary to create the nonoxidizing environment, for instance, by using the electrolyte sparged with helium.30a The same approach was found suitable for protecting sulfide and oxysulfur species against oxidation during separation.37 Chromate, which is the most commonly used separation electrolyte co-ion for sulfur-containing anions,37b,38 was shown to be inappropriate in some cases (e.g., for sulfite,38a,e metabisulfite,39 or dithio-

3.2. Approaches for Improving Resolution

In a few instances, the different chemical forms of a particular element are nicely separated due to sufficient variation in the mobilities as a result of differences in the charge or size of the species (or both). However, a more frequent occurrence is when precapillary or in situ (in-capillary) transformation of the analytes, a special adjustment in the electrolyte composition, or a change of separation hardware is required to enhance the resolution. This is especially true if an element is present in different states with varying concentrations. In the following section, the significant amount of work carried out in recent years in developing CE systems to ensure that the target analyte 781

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species arrive at the detector well-separated from each other is presented in detail. 3.2.1. Species Derivatization. The specific chemical derivative transformation of analyte species is a powerful separation strategy used so that careful control is undertaken in order that speciation does not undergo any change. For the determination of different metal charge states using CE separation, derivatization using a strongly complexing reagent into a single distinct complex with each species3b remains the most viable option. In this way, the different redox forms of chromium42c,e,54 or vanadium42e,55 attain the same (negative) net charge (and also high molar absorptivities) by converting both the Cr(III) and vanadium species into chelate (or Keggin type55a) complexes. This is achieved before initiating the separation. However, most of these complexing reactions are fairly slow and require heating. The latter process may result in significant conversion of Cr(VI) into Cr(III).50a In addition, for Cr(III) more than one kind of complex might be formed.50a In this context, a number of approaches have been used to separate the chromium species directly, without derivatization.56 These are well-documented and include using acidic electrolytes and pressure-assisted electrophoresis,57 shorter separation pathways,57b,c,58 or, less advantageously, two separate electrophoretic runs50 or off-line detection.59 A more efficient CE methodology, also requiring no species transformation, is based on the dual opposite-end injection.60 The sample, containing both cationic and anionic Cr species, is introduced into the opposite ends of the capillary, and after a high voltage is applied, the analytes migrate toward the capillary center, where the detection cell is placed. Typical electropherograms are shown in Figure 1. The same element speciation was also directly analyzed in one of very few contributions exploiting capillary electrochromatography (CEC). Here, the separation mechanism is governed by a combined action of anion complexation and electrophoretic movement.61 Given sufficiently rapid complexation between the metal analytes and the ligand, an in-capillary complexation mode might be a judicious choice, since it involves minimal sample dilution. This proved to be the case in several instances, for example, the V(IV)/V(V) separation using an EDTAcontaining electrolyte62 or the differentiation between Co(II) and Co(III) or Cr(III) and Cr(VI) using 1,10-phenanthroline (o-phen)63 or 1,5-diphenylcarbazide,64 respectively. These were introduced into the capillary either before or after the sample and then reacted with the separated analytes (so-called zonepassing technique). In the latter case, chromium(III) was also in-capillary oxidized by Ce(IV). To separate Fe(II) and Fe(III), a common approach is to adjust their effective mobility by conversion into oppositely charged complexes.42b,e,65 This can be achieved by employing two complexing agents, o-phen and EDTA (or trans-1,2cyclohexanediaminetetraacetic acid) (added to the sample42b,e,66 or running electrolyte65b), transforming the iron species into [Fe(o-phen)3]2+ and [Fe(EDTA)]−, respectively. To prevent metal chelate dissociation during the CE separation, o-phen and EDTA were also added to the electrolyte (see also section 3.1). Another research group achieved the separation of iron oxidation states in the form of anionic complexes using incapillary complexation with 2,6-pyridinedicarboxylic acid (PDCA).67 It is noteworthy that due to complexation the redox potential of Fe(III)/Fe(II) may increase, thus preserving the stability of the iron species in the sample solution or electrolyte.

Figure 1. Simultaneous determination of Cr(III) and Cr(VI) in (a) standard solution and (b) rinsewater from the galvanic industry. Sample treatment: dilution (1:1000). CE conditions: capillary, fusedsilica, 50 μm × 50 cm; electrolyte, 4.5 mM L-histidine adjusted to pH 3.4 with 10% v/v acetic acid; sample introduction, hydrostatic, from a height of 20 cm for 30 s (cathodic end) and 30 s (anodic end); time between the injections, 15 s; voltage, 17.5 kV; detection, CCD. Reprinted with permission from ref 60. Copyright 2003 Wiley-VCH Verlag GmbH & Co.

Organometallic compounds only slightly dissociate in an aqueous medium and often possess similar migration velocities within a single metal family. Hence certain adjustments are required in order to improve the resolving power of the CE system. For instance, separation between different cationic organomercurials, which is usually rather poor due to low or no charge, experiences a handsome enhancement after precapillary or on-capillary derivatization. This commonly involves cysteine68−70 or a few other mercapto reagents.68f,71 Alternatively, nonaqueous CE, with a buffering component dissolved in acetic acid72 or methanol,73 can be utilized. Little guidance is available in the literature for making use of the derivatization principle in separating anionic species.74 The one-to-one association between divalent cationic hexamethonium [1,6-bis(trimethylammonium)hexane] and perchlorate and other chlorine oxide anions results in complexes with a net positive charge, which facilitates their separation.74a It has been reported that in-capillary complexation with an azacryptand, which is stronger for nitrate than nitrite, led to their peaks emerging in reverse order and at shorter migration times.74c One specific type of analyte that does require derivatization is that of the seleno amino acids that contain an asymmetric α782

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carbon atom.75 In order to discriminate between the D- and Lenantiomers, they need to be derivatized into diastereomers using a chiral selector such as 1-fluoro-2,4-dinitrophenyl-5-Lalaninamide.75b Alternatively, an electrolyte containing the selector can be used, as demonstrated by separating all enantiomers of three seleno amino acids using sulfated βcyclodextrin and vancomycin additives.75a In another example, a mixed micellar system of chiral taurodeoxycholic acid and achiral SDS surfactants along with β-cyclodextrins was employed.76 3.2.2. Adjustment of Electrolyte Composition. The pH presents a significant electrolyte variable that can be used to control the separation selectivity for pH-sensitive species. To enable the fast separation of the individual oxoanions of arsenic,34b,77,78 selenium,79 or both metalloids,51,80 along with their organic forms, the pH should be adjusted to pH ≥9. At this pH, all the species are essentially ionized. Similarly, the baseline separation of six selenium species81 and five phenylarsenic compounds82 was only feasible after a thoughtful alteration of the pH of the running electrolyte to 8.4 and 8.0, respectively. On the other hand, the use of acidic electrolytes can lead to enhanced selectivity for nitrate and nitrite,74c,83,84 since the latter dissociates only incompletely at low pH. The most advanced approach, based on electrokinetic sample injection from both ends of the capillary,83b,c led to their simultaneous separation from ammonium ions. In addition, at pHs below 3, capillary surface silanol groups become fully protonated and this gives rise to the electroosmotic flow (EOF) being directed toward the anode (see also the following subsection). Other approaches to regulating electrolyte composition in order to complete the resolution of complex mixtures or analytes that differ only slightly in electrophoretic mobility include the following: (i) varying the nature of the electrolyte buffer,29a (ii) increasing the concentration of the counterion85 (to evolve ion association with the separand), and (iii) addition of an organic solvent such as methanol,24c,29a,b,52,69a−e,70c,78b,86 acetonitrile,38d,42c,87 ethanol,88 or 1-butanol.86c In the case of hydrophobic or zwitterionic analytes, micellar,34b,c,65a,68c,86a,89 nonaqueous,72,74a,90 or cyclodextrin-modified68e,91 electrolytes can all be utilized for improving separability. Research has also been devoted to the enantioselective separation of metal− ligand complexes using electrolytes containing chiral additives, such as various tartrate derivatives,92 cyclodextrins,93 synthetic DNA polynucleotide,92c sodium cholate94 or other bile salt surfactants.94a Other examples are based on a cationic polymer95 or a microemulsion.96 It is important to point out that using the charged surfactants at levels above their critical micelle concentration moves the separation mechanism into the realm of micellar electrokinetic chromatography (MEKC). Under some circumstances the use of surfactants is undesirable (e.g., because of their reduced compatibility with ESI-MS). The implementation of electrochemically assisted injection, which enables the generation of charged species from neutral analytes, such as various ferrocene species,90a,97 could prove to be an alternative approach. However, this technology is not easily accessible in common analytical laboratories, which of course impedes its wider application. Another practical precaution involved with the use of coupled detection systems, which are described in detail below in section 4, is due to the fact that a part of the capillary remains outside the CE instrument and hence is not cooled. This imposes the condition that the

electrolyte concentration needs to be kept fairly low in order to maintain the separation efficiency.30a 3.2.3. Modification of the Electroosmotic Flow. The reversal of the EOF is desirable in order to promote the migration of anionic species in the same direction as the EOF; this is called the coelectroosmotic mode. It ensures that all species are moving to the detection end of the capillary and thus reduces the analysis time. Most commonly, anodic EOF is created by employing a cationic surfactant. Typically, a quaternary ammonium compound, such as cetyltrimethylammonium chloride (CTAC),84a,c,98 hydroxide,77g,78d,79a,b or bromide,38c,49,77a,79d,81,99 is introduced as an additive to the running electrolyte used. Others that have proven useful are tetradecyltrimethylammonium bromide39,42c,55b,67,80b,87b,c,100 or hydroxide,37b,38d,54a,b,83b,c,101 dodecyltrimethylammonium bromide,41a,102 hexamethonium bromide38f,103 or hydroxide,38c,99c octadecyltrimethylammonium hydroxide,80a etc.78a,104 It should be noted that in general these EOF modifiers are not always inert with respect to the anionic analytes.38c,49,105 By interacting with them, they may induce not only alterations in the resolution (by an ion-exchange mechanism) but also compromise the separation efficiency, cause signal suppression, and even interfere with some detection modes. Therefore, amines can be used instead of the surfactants. A typical example is diethylenetriamine,38a,e which does not reverse but suppresses the EOF. Cationic polymers allow greater flexibility in optimizing the separation conditions.42e An alternative possibility is to use the EOF modifiers not by adding them to the electrolyte but rather by conditioning the capillary with a surfactant/polymer solution in a number of ways. This methodology can be used prior to each run,32,83a,e,106 after a number of runs,77c,88 such as every 10−100 analyses or even longer,86d,107 or by combining an overnight flushing with the addition of a polymer to the electrolyte.108 In each of these cases, the modifier dynamically or semipermanently covers the inner surface of the capillary and changes its charge from negative to positive. For instance, when the capillary was pretreated with a Polybrene (hexadimethrine bromide) solution, fluoroacetate and its serum metabolite, fluoride, were successfully separated in about 4 min.109 On the other hand, when chitosan was employed as the dynamic EOF modifier, it became possible to carry out separations of saliva nitrogen-containing anions in less than 20 s. It should be noted that this was also due to the use of the short-end-capillary injection technique.53 An even more stable and permanent coating can be realized for the capillary treatment with the use of a cationic polymer such as polyethyleneimine 2 8 , 3 2 or poly(diallyldimethylammonium chloride).80c Both of these are inclined to be irreversibly adsorbed onto the capillary wall. In a similar approach, good stability of the capillary coating was achieved as a result of applying successive multiple ionic polymer layers; the first and the last of these layers was Polybrene-based.78c Rinsing with a Polybrene solution prior to each analysis additionally protects the coated capillary against any performance deterioration.110 However, a recognized drawback of multiple coatings, which makes it a much less frequently used strategy, is that the method may suffer from nonreproducibility. A search of the literature reveals a few contributions using chemically modified capillaries to attain rather high and virtually pH-independent EOF for separating chlorine-111 or nitrogen-containing anions112 and different metal−protein species.32,113 The chemistry involved in capillary 783

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Figure 2. Separation of nitrogen-containing anions in human saliva using (A) CTAC (1 mM) and (B) Zwittergent-3-14 (2.5 mM) additives. CE conditions: capillary, fused-silica, 50 μm × 44 cm; electrolyte, 10 mM phosphate buffer (pH 7.0) containing the respective additive; sample introduction, pressure for 4 s (sample volume 40 nL); voltage, −30 kV; detection, UV (215 nm). Peaks: (1) NO2−, (2) NO3−, and (3) SCN−. The lower traces shows electropherograms obtained after 10 consecutive runs. Reprinted with permission from ref 124. Copyright 2001 Springer Verlag GmbH & Co.

demonstrating the advantages of combined CEC separations over any single-mechanism system and would-be applications. This is apparently due to challenges in producing suitable columns. 3.2.5. Amendments for Real-Sample Analysis. A CE separation developed using a standard mixture may often exhibit difficulties when the method is applied to a real sample. These include large variations in migration times between standards and samples, nonreproducibility with respect to migration times or peak shapes, poorly resolved electropherograms, baseline shifts, the occurrence of system peaks, and even the appearance of unknown species of the target element.34b The phenomena of shifts in migration times and peak broadening are attributed to the difference in conductivity (ionic strength) of the sample with respect to the electrolyte (electromigration dispersion) and standard solutions. Furthermore, if the conductivity of the sample is considerably higher compared to that of the electrolyte, the sample ions will experience a lack of electric field, and as a result, the separation will be degraded and even cease because of electrical breakdown. As in the case of dealing with alleviating species instability, the most effective and straightforward strategy for overcoming these problems is to modify the electrolyte so that it matches the sample matrix with respect to both the nature and concentration of the dominant ion(s). The success of this strategy has been repeatedly demonstrated in cases quantifying anionic forms of nitrogen and iodine in seawater by employing artificial seawater83e,98a,121 or 0.5−1.5 M NaCl121e,122 as the carrier solution. Under these conditions, the EOF is greatly reduced, and this eliminates the need for an EOF modifier. In some of the above reports, CTAC was added to the separation electrolyte, but its role was to serve to adjust the effective mobility of certain analytes by virtue of specific ion association. Likewise, electrolytes similar in composition to protein-free serum have proven useful for the simultaneous determination of NO2− and NO3− in this particular biofluid.19a,20,98b,c Using

coating results in the formation of a covalently bonded zwitterionic salt with sulfonic acid groups or a sulfonated polymeric layer which shows improved, faster separations and reduced adsorption effects compared to either unmodified fused-silica capillary systems or capillaries coated dynamically with cationic materials.32 Nonchemical ways to amend the EOF by applying a hydrostatic pressure opposite to the direction of the EOF69a or an appropriate suction force from the nebulizer of an ICPMS interface114 were also proposed with the aim of improving the resolution of mercury and antimony species, respectively. 3.2.4. Use of Supplementary Separation Mechanism. It would be incorrect not to mention early work that made use of packed and open-tubular capillaries in order to manipulate separation selectivity by incorporating a chromatographic principle into the CE system. The suitability of using short columns for a number of relevant analytes has been investigated. Among these are a column packed with a polymer-based115 or a silica-based116 anion-exchange (AE) material; a histidine-functionalized silica converted into the Cu(II)−histidine complex, which apparently acts as a ligand exchanger;117 fused-silica capillaries coated with commercial AE latex particles, 116,118 a macrocyclic polyamine, 61 or a diazacrown ether;119 and, finally, a cationic polymer120 possessing anionic selectivity. The analytes studied include common inorganic forms of iodine, bromine, chlorine, and nitrogen,115−120 as well as a series of As, Cr, and Se species.61 The zwitterionic character of a commercially coated ZICcapillary makes it a reasonable candidate for chiral separations.87d It consists of a silica capillary with covalently attached sulfobetaine-type functionalities and it offers hydrophilic interactions (with a high percentage of acetonitrile in the electrolyte) suitable for resolving the diastereomers of a Fe(III) complex. Nonetheless, research efforts on combining chromatography and CE mechanisms have not really progressed much beyond 784

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characterizing of Zn(II) complexes with carbonic anhydrases from red blood cells22 and quantifying the protein adducts of ruthenium(III)-based drugs,126b,c respectively. Similarly, polyacrylamide-coated capillaries have paved the way in attaining the goal of clarifying the properties of MT isoforms,91,129 including those existing in liver or pancreas cytosol,24a,b,e,f,35,130 without the risk of nonspecific adsorption of the analytes. Even simpler modifications to CE systems, such as using a low-pH electrolyte, may occasionally be helpful in eliminating the interference from matrix components and help to achieve the electrophoretic purity of the analytes reaching the detector. In such electrolytes, carbonate, which typically is excessive in water or soil samples, turns out to be uncharged and migrates with the EOF.131 Other researchers have increased the pH of the electrolyte buffer in order to tackle the problem of comigrating anions from soil solutions.104b A noncomplexing electrolyte based on calcium chloride132 could help to successfully analyze samples with unknown matrices for which derivatization can be occasionally incomplete. If none of the strategies described prove helpful, the suitability of the sample preparation procedure should be checked (see section 5).

electrolyte buffers as concentrated as possible is another competent strategy for the challenge of making the electrophoretic transport of the analytes less sensitive toward matrix effects such as pH extremes and impurities among others. Alternatively, in order to restrain the resolution from being affected by a troublesome sample, the electrolyte composition can be further adapted by incorporating a suitable additive. A good example is the case where an electrolyte, which performed well in resolving four selenium species as the standards, had in practice to be subjected to virtually complete compositional change when applied to urine analysis.79b Addition of a zwitterionic surfactant [e.g., 3-(N,N-dimethyldodecylammonio)-propane sulfonate105 or N-tetradecyl-N,N-dimethyl-3ammonio-1-propanesulfonate123] altered the migration order of the analyte anions by acting as a pseudostationary phase and introducing a chromatographic component into the separation. In effect, this removed them from the large chloride matrix peak, thus making it feasible to carry out the determination of NO2− and NO3− in seawater.105 In another example, by applying a mixture of zwitterionic and nonionic surfactants, this permitted the use of high ionic strength electrolytes for the analyses of undiluted seawater without the occurrence of any significant peak broadening.123 Similarly, when sulfobetainetype zwitterionic micelles were used as the separation medium, this strategy enabled the direct determination of three biologically important nitrogen-containing anions in human saliva (Figure 2). As can be seen from a comparison of the electropherograms in the figure, this kind of biospeciation analysis has the advantage of preventing adsorption of the sample proteins onto the inner wall of the capillary by using the zwitterionic additive. A similar function with regard to the same analytes/sample is played by using an uncharged hydroxypropylcellulose polymer that is introduced into the separation electrolyte.124 Another zwitterionic electrolyte additive, Ncyclohexyl-2-aminoethanesulfonic acid, was utilized to reduce the adsorption of proteins in the direct assay of nitrite and nitrate in human plasma; that is without the need for deproteinization.20 Matrix proteins or other endogenous substances can precipitate on contact with a cationic surfactant used as an EOF modifier. To circumvent this undesirable effect, the dynamic coating of the fused-silica capillary with a charged polymer holds great promise in biospeciation analysis.126 A number of selenium compounds occurring in aqueous extracts of selenized yeast were found not to be baseline separated, and even after 30 min, 25−30% of the selenium content did not leave the untreated capillary.79d Coating the capillary with poly(vinyl sulfonate) proved beneficial. The results provided a fingerprint portrait of selenized yeast preparations, with more than 20 selenium species separated within 13 min127 (unless this is partially due to artifacts caused by the capillary coating). The use of chitosan and glutaraldehyde was shown to prevent the adsorption of proteins present in saliva samples. This strategy also simplifies capillary conditioning between runs and minimizes sample pretreatment.53 A low molecular mass compound, spermine [N,N′-bis(3-aminopropyl)-1,4-butanediamine], that does not precipitate proteins allowed direct CE analysis of airway surface liquid.128 When analyzing the constituents in blood samples, protein adsorption onto the surface of uncoated capillary walls can also present overwhelming challenges. The use of capillaries either dynamically or semipermanently coated with Polybrene was proposed as a means to eliminate this problem. This approach was used in the

4. DETECTION SYSTEMS 4.1. Couplings to Element-Selective Techniques

It is clear that the identification and determination of different elemental species using CE techniques combined with an element-specific detector make speciation analysis more reliable, not to mention improving the sensitivity. This fact has been recognized by those involved in the development and commercialization of CE systems. Of the several options for online hyphenation with CE, ICP-MS has received particular attention,6a,133 and hence, it is considered first in this section. 4.1.1. Inductively Coupled Plasma Mass Spectrometry. ICP-MS provides the advantage of extremely low detection limits and as a detection technique coupled to CE alone possesses a sensitivity potential capable of resolving the challenge of quantifying trace species. Compared with other element-selective methods, ICP-MS is unique in possessing the attractive assets of multielemental and even multi-isotopic detection capabilities. Importantly, if an ICP-MS detector is used, the necessity to electrophoretically separate species of different elements from one another no longer arises. This is because their electropherograms can be recorded separately with no interelement interference.34e,42a,80b,114a Furthermore, the resolution can be traded off against analysis speed, since species with similar mobilities do not have to be separated. As a consequence, there are greater possibilities for the characterization and quantification of multispecies systems. Additionally, there is an increased freedom in choosing the separation electrolyte that should prevent loss in speciation information. On the other hand, it should be noted that any electrolyte component, including the buffer, micellar agent, salt, complexing agent, etc., is not expected to interfere with the ICP measurement. For this reason, and also to avoid the clogging of the nebulizer, the concentration of the electrolyte should be kept as low as possible. Alternatively, special additives, such as a nonionic surfactant,40 can be employed. The coupling of CE with ICP-MS, however, requires an interface to match the flow rate of the solution coming off the capillary (μL min−1) with the vastly different uptake rate needed for the stable operation of the ICP (mL min−1). In 785

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Figure 3. Manganese-specific electropherogram of human cerebrospinal fluid sample detected by ICP-DRC-MS. CE conditions: capillary, fusedsilica, 50 μm × 120 cm; electrolyte, 200 mM sodium acetate buffer, pH 6.2; sample introduction, pressure, 345 kPa s; voltage, −18 kV; detection, ICP-MS (m/z 55). Peak assignment: (2) Mn−histidine, (3) Mn−fumarate, (4) Mn−malate, (5) Mn−oxalacetate, (6) Mn−α-ketoglutarate, (7) Mn−nicotinamide-dinucleotide, (8) Mn−citrate, (9) Mn−adenosine, (13) inorganic Mn; other peaks not identified. Reprinted with permission from ref 143a. Copyright 2007 Wiley-VCH Verlag GmbH & Co.

dilution factors in sheath-flow interfaces set limits on attaining inherently very high ICP-MS sensitivity, they can be considered an advantage from the viewpoint of preventing some matrixrelated interferences. When using quadrupole ICP-MS, the determination of certain elements can be difficult due to a low degree of ionization in the argon plasma or monoisotopy, but this can be even more problematic in the case of mass spectral interferences from polyatomic ions that reduce the ICP performance. This can be overcome by the implementation of either dynamic reaction cell (DRC) or collision cell (CC) methodology. The first approach is based on a chemical reaction with reactive gases in order to create product ions that cause less interference. For instance, the determination of vanadium, chromium, and iron (at m/z 51, 52, and 56, respectively) in wastewater was greatly facilitated by alleviating any interference from 35Cl16O+, 40Ar12C+, and 40Ar16O+ ions by the use of an ICP-MS instrument equipped with a DRC in which ammonia functioned as the reaction gas.42e Likewise, the isobaric interferences of 40Ar38Ar+ and 40Ar40Ar+ in the detection of 78Se and 80Se were completely eliminated using hydrogen99g or methane51 as the reaction cell gas. The result was significantly reduced DLs. When CH4 is employed, the simultaneous determination of arsenic species is possible in the form of the adduct ion 75Ar12CH2+. Moreover, the addition of methanol to the sample solution brought about higher signal intensity.99g This is an effect of an increased population of carbon-containing ions in the plasma, which in turn supports the ionization of selenium. Even without using this approach, the selenium speciation can be analyzed by employing a DRC with sensitivity comparable with that of CE-sector-field (SF)− ICP-MS.142 This is discussed below. Using methane as the reaction gas, 13 Mn species were detected in cerebrospinal fluid.143 Of these, nine have been identified in spite of the fact that their signals are present only at LD levels (Figure 3). The effect of the reaction with O2 in the DRC on the detection of sulfur-containing amino acids is clearly seen from a comparison of the sulfur-selective electropherograms shown in Figure 4. A significant difference in the signal count rates is due to analyte detection in the form of 32S16O+ and 34S+ (having a low natural abundance) in the DRC and standard mode, respectively.

addition, the interface should maintain a steady electrical connection to the CE system as well as making it possible to efficiently introduce analytes into the plasma.134 Degradation of the resolution and excessive dilution are to be avoided. Basically, there are three types of interfacing systems that can be used to combine CE with ICP-MS: a sheath-flow interface, a nonsheath-flow interface, and the hydride generation (HG) interface. Of these the former has received the most attention. Furthermore, with the recent advent of commercial sheath-flow interfaces, which are based on a total-consumption nebulizer with a miniaturized low-dead-volume spray chamber and optimized fluid dynamics, the technical problems of interfacing the CE separation system with the ICP-MS detection apparatus have been overcome. This spurred growth regarding the range of possible of CE−ICP-MS applications. It also ensured its competitiveness with respect to HPLC, a general-purpose method in the field of speciation analysis. The early work on developing high-efficiency nebulizer systems, started around 1995 with the first laboratory-made constructions, is not considered here, but the interested reader is referred to the abundant review literature.6a,14a,134,135 However, the issue regarding the considerable dilution of analytes in commercial interfaces (often by a factor of >100), which limits the detection sensitivity, impels further efforts toward improving the interface design. One such interface with greatly reduced dead volume (