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Capillary electrophoresis inductively coupled plasma mass spectrometry
Microchemical Journal 66 Ž2000. 3᎐16
Capillary electrophoresis inductively coupled plasma mass spectrometry Vahid MajidiU Analytical Chemistry Sciences, MS K484, Chemical Science and Technology Di¨ ision, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
Abstract Chemical speciation Žextraction of elemental information and identification of molecular environment for an analyte in a complex sample. has been a long sought after goal for analytical chemists. Recently, because of successful developments in more sensitive element-specific detectors and gentle separation schemes, which preserve the true chemical information in a real sample, routine speciation experiments are becoming a common occurrence in the scientific literature. For many reasons, the combination of capillary electrophoresis Žfor separation of different chemical species. with inductively coupled plasma mass spectrometry Žfor element and isotope specific detection. has emerged as the method of choice for these analyses. In this article the basic principles of capillary electrophoresis inductively coupled plasma mass spectrometry are discussed. Design consideration for instrument interface, anticipated difficulties with speciation experiments and applications for specific matrices and analytes are also presented in this article. 䊚 2000 Published by Elsevier Science B.V. Keywords: Capillary electrophoresis; Inductively coupled plasma mass spectrometry
1. Introduction Continuing technological development in separation science and spectrochemical detection has ultimately resulted in evolutionary improvements in hyphenated and tandem techniques. Combination of two or more analytical techniques always
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provides a higher data density than either technique individually. This has been the prevalent trend in instrument development research during the past decade. In the 1980s, the spectroscopy and the separation science communities were witness to a parallel development and maturation of two powerful instrumental techniques: capillary electrophoresis ŽCE. and inductively coupled plasma mass spectrometry ŽICP-MS.. Capillary electrophoresis became commercially available in the latter part of
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the 1980s and rapidly emerged as a routine tool for many clinical and pharmaceutical applications. In reality, capillary electrophoresis is a family of separation techniques based on electrophoretic migration of analytes in a capillary w1x. Among the positive attributes associated with CE, perhaps the most important advantage with regard to chemical speciation applications is the lack of a stationary phase in the separation column. When dealing with kinetically or thermodynamically labile molecules, the analyte interactions with a stationary phase in conventional chromatographic separation techniques may be detrimental to the analysis. That is, the nature of the analytes introduced onto the separation column may be altered because of their interactions with the stationary phase. In CE methods, the electrophoretic forces drive the separation and as such there is no need for a stationary phase to impose specific chemical interactions. In one case, electrochromatography, a packed capillary is used to allow for distinction between similar compounds. Nevertheless, capillary electrochromatography does not really belong to the CE family and the electrical potential imposed across the column is merely a ‘pump’ to generate an electroosmotic flow through the column. Another significant analytical development during the 1980s was the invention and commercialization of inductively coupled plasma mass spectrometry, first described by Houk et al. w2x. This hybrid instrument exploits the energetic plasma in an ICP to completely destroy the sample matrix and efficiently ionize the constituent elements. When an ICP source is combined with mass spectrometric detection, one of the most sensitive element-specific detectors is obtained Žbecause the majority of the elements passing through the plasma are nearly completely ionized.. ICP-MS can provide quantitative elemental and isotopic information with a dynamic range exceeding five orders of magnitude and instrumental limits of detection better than 1 ngrl for most elements w3x. Although this instrument can provide nearly simultaneous multi-elemental analysis, because of the energetic nature of inductive plasma, the speciation information is not preserved.
Extraction of elemental speciation information from small samples or dilute analytes is best achieved through the on-line combination of ICPMS with an instrument capable of separating analytes based on their chemical w4,5x or physical properties w6x. The readers interested in coupled techniques for chemical speciation will find the comprehensive review articles by Lobinski w7x, Sutton et al. w8x and Vanhaecke and Moens w9x most useful. From the list of potential coupled techniques, the combination of CE with ICP-MS seems to provide a set of performance characteristics unmatched by any other technique. Perhaps, the second most versatile detection approach for CE is the electrospray technique. Unfortunately, not every bufferranalyte combination is amenable to analysis by the electrospray technique, the detection limits are not as good as ICP-MS and the linear dynamic range is extremely limited. These two approaches should not be considered as competitive but rather complementary techniques. Houk w10x, Lobinski et al. w11x and Michalke et al. w12x have reported comparison of these two techniques for various types of analysis. It is not surprising that the potential utility of CE in tandem with ICP-MS was recognized relatively early in the speciation community. However, perceived difficulties in designing a functional interface for coupling of CE with ICP-MS discouraged most researchers from this venture. The operational parameters for CE and ICP-MS experiments were individually well established. However, these parameters are not necessarily compatible between the two techniques. For example, the flow rate of run buffer in a CE column can be as little as a few nlrmin, while the conventional ICP-MS sample introduction requires a flow rate of approximately 1 mlrmin. The requirements for electrical continuity along with six orders of magnitude difference in flow rates between the two systems were the main reason for the postponement of an interface development to the mid-1990s. It is fitting that Olesik et al. w13x reported the first functional interface for CE-ICP-MS. Olesik w14,15x had previously worked specifically on evaluating the sample uptake, aerosol formation, aerosol transport, and the atomization mechanisms in both ICP and ICP-MS.
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Armed with this knowledge, Olesik understood that the ICP-MS could operate effectively with smaller sample flow rates and that at these small flow rates Žlrmin. the aerosol formation and transport are very efficient. Using this basic concept and a small make-up flow, a successful CEICP-MS coupling was achieved. Today, almost every combination of nebulizers and spray chambers has been exploited for CE-ICP-MS applications. In a brief review, Barnes w16x describes a general overview of CE-ICP-MS technique, instrumental requirement and the potential for microfabrication. Interested readers may also find the manuscript on use of ultrasonic nebulizers for CE-ICP-MS applications, by Kirlew et al. w17x, relevant to the content of this article.
2. Instrumentation It is not the intention of this article to fully cover the instrumental details associated with CE or ICP-MS. Several excellent books with specific focus on each specific technique are available on these subjects w1,3x. Our goal is to present a brief overview of these techniques so that the difficulties associated with using them in a tandem arrangement will be apparent and the solutions used to deal with these issues can be better appreciated. 2.1. CE Capillary electrophoresis is a simple and powerful separation technique. The separation is often achieved in a fused silica capillary with an internal diameter of 25᎐100 m. The instrument is completely operational when the capillary is filled with a buffer and both ends of the column are submerged in two different buffer reservoirs equipped with electrical connections. Platinum electrodes are used to establish a potential difference between the two ends of the capillary. A potential difference of up to 30 kV is applied to the 1-m-long capillary, which results in electric fields of up to 300 Vrcm. Typically, the maximum potential is selected so that the run currents are maintained below 100 A. The general schematic
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Fig. 1. A block diagram for conventional capillary electrophoresis instrument with an UV detector.
arrangement of a CE instrument is shown in Fig. 1. In CE the absolute potential difference between the two end of the capillary is not, by itself, a critical parameter. However, the electric field imposed on the capillary Žpotential difference divided by the length of the capillary. ultimately determines the resolution and separation efficiency. Parameters that profoundly influence the CE separation include the types of ions present, the internal diameter of the capillary, buffer ionic strength and pH. In most instances, these factors work in concert to effect the separation characteristics. For example, by increasing the ionic strength of the buffer, better resolution is often expected. As the ionic strength of the solution is increased the temperature of the run buffer becomes higher because of increased current for a given potential difference Žresistive heating.. This in turn leads to better thermal diffusion of analytes resulting in an exacerbated resolution. Use of smaller diameter capillaries will allow for reduced run currents and a more efficient cooling of the capillary. These interdependencies are not a mere linear function of one another, and subsequently, optimized run conditions have to be established for a unique combination of buffer, capillary and applied electric field. Fortunately, most of these combined parameters have been evaluated and presented in the literature. If a unique set of parameters is being used, as expected for application of CE in tandem techniques, an Ohm-law plot should be constructed to avoid some unacceptable experimental conditions. The Ohm-law plot simply shows the depen-
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dency of the observed run current as a function of applied potential across the capillary. Ideally, a linear relationship between the applied potential and the measured current should be obtained Ž V s IR, where V is the applied potential, I is the measured current and R is the resistance of the buffer solution within the column.. At higher potentials, however, this linear response may not be observed as depicted in Fig. 2. The best run condition is commonly selected at the highest possible potential within the linear region of the Ohm-law plot. In a gross oversimplification we can state that the best separation conditions are obtained in capillaries with very small diameters, at high ionic strengths and large imposed electric fields.
Fig. 2. A plot of measured and expected current in a CE column as a function of applied potential.
2.2. ICP-MS In contrast to CE, ICP-MS is a highly complex instrument that relies on two decades of technology development in RF plasmas and ion optics for mass spectrometric detection. Today’s instruments are highly automated based on a series of predetermined experimental parameters programmed by manufacturers. To some extent, this high level of automation is a nuisance for tandem instrumental applications because the optimized parameters for the operation of the ICP-MS in a hyphenated mode may not be similar to the conditions optimized for ICP-MS alone. As we mentioned previously, detailed description of ICP-MS instrumentation can be found elsewhere w3x. Briefly, this instrument consists of a plasma torch ion source and a mass analyzer. The plasma is generated in an Ar environment by a high-power radio-frequency generator Žapprox. 1 kW.. The resulting plasma possesses high excitation and ionization temperature. The species traveling through this plasma are atomized and ionized with a very high efficiency. The ions are extracted from the ICP torch into a mass analyzer through a two-stage, differentially pumped, sampling and skimmer cone assembly. The sampled ions are directed toward the mass analyzer Žoften a quadrupole mass filter. with a series of ion optics assemblies. A schematic diagram of the ICP-MS is shown in Fig. 3.
Fig. 3. A simplified schematic representation of an ICP-MS instrument.
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2.3. Interface The interface can be best represented as a combination of three subsystems: spray chamber, nebulizer and the CE connection hardware. While the manuscripts published on this topic often contain the phrase ‘a new interface’ Že.g. Schaumloffel and Prange w18x. in their corresponding titles, in reality they refer to an alternative combination of CE-connection with different nebulizerrspray chamber arrangement. Nonetheless, Lu and Barnes w19x, Tangen et al. w20x and Taylor et al. w21x have described some innovative combinations of the above three components for CE-ICP-MS instrumentation. The basic CE-connection hardware to any nebulizer is virtually the same in all publications with only minor variations in some instances w22,23x. The schematic of this generalized CE-connection to a generic nebulizer is shown in Fig. 4. As shown in the figure, the separation capillary is inserted through a Peek tee Žmodel P-728. and it is held in place using a model F-300 Peek fitting and a 0.4-mm Vespel ferrule ŽAlltech Associates Inc., Deerfield, IL, USA.. All tees, fittings, PEEK ferrules, and Teflon tubing were purchased from Upchurch Scientific ŽOak Harbor, WA, USA.. The sheath buffer flow is mixed with the CE effluent prior to nebulization and the mixing of the buffer solutions occurs at the tip of the separation capillary Žthe buffer sheath flow is also the electrical connection for the circuit continuity.. Teflon tubing Ž0.76 mm i.d.=1.6 mm o.d.. attached to a peristaltic pump supplies the buffer flow to the interface. The cathodic electrical connection is made through the same make-up buffer port of the interface tee by placing a 0.076 mm o.d. platinum wire between the Teflon tubing and the Peek ferrule. Another 0.76 mm i.d. Teflon tubing is used to guide the buffer sheath flow and the separation capillary into the tip of the nebulizer. The above CE connection can be attached to any nebulizer to facilitate analyte transport into the ICP torch. Some of the most commonly used combination of nebulizers and spray chambers is shown in Fig. 5. These combinations of CErnebulizerrspray chamber are all functional, and their performance mostly depends on the
Fig. 4. The general interface design for capillary electrophoresis.
specific goals for each analysis. In some instances the performance of these interfaces have been handicapped because of the self-aspirating nature of the commercial pneumatic nebulizers. If not regulated, the aspiration in these nebulizers causes a significant laminar flow within the capillary. Kinzer et al. w24x has discussed the consequence of this laminar flow. The aspiration rate of each nebulizer is a function of the Ar gas flow rate and the nebulizer design. The data shows that an increase in the nebulizer gas flow rate will result in faster migration velocities of analytes within the capillary. The negative pressure at the inlet of the nebulizer causes this suction of the analyte in the capillary. Nonetheless, this problem can be easily resolved or minimized with judicious use of makeup buffer flow and nebulizer gas flow rates. In general, when attempting to use the commercial nebulizerrspray systems for CE applications, one has to consider the parameters that influence the separation efficiencies as well
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detection limits. Total dead volumes, composition of buffers and flow compatibilities are also among the parameters that must be optimized for a successful union of CE with ICP-MS system. 2.4. Unique approaches Stability and ease of fabrication of the above simple interface is responsible for its vast popularity. Other unique interfaces have been designed for transport of CE effluent into the ICP torch, but with limited success. One successful design, by Chan and Chan w25x, uses an integrated approach for analyte sampling. In this design, the capillary is made of two segments connected by a low dead-volume tee. This tee provides the electrical continuity for the CE separation as well as being the outflow channel toward the ICP torch.
Another design promoted by Mei et al. w26x completely eliminates the need for a makeup buffer and the electrical connection is made directly through a thin Pt wire inserted into the cathodic end of the capillary. In either of the above cases the control and optimization of separation and analyte introduction parameters is non-trivial. Tian et al. w27x published a clever concept for connection of CE to an ICP torch. Recognizing that hydride generation can significantly improve the detection limits for hydride forming elements, they designed an interface based on a movable reduction bed hydride generation system. The advantages of such system include excellent analyte transport and some analyte pre-speciation based on hydride formation selectivity. Because of the hydride generation step, this system cannot be used with all elements.
Fig. 5. Different nebulizers and spray chamber configurations typically used for CE-ICP-MS.
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Considering that the sample loading in CE is typically less than 100 nl, the major drawback, when using CE for chemical speciation, is the limited amount of sample utilized for the analysis. Recognizing this limitation, Majidi et al. w28x published a unique approach, which takes advantage of parallel separation scheme to realistically improve the analytical sensitivity and detection limits. Several individual capillaries with identical internal diameters and lengths were placed in a loose bundle and because the capillaries were not physically in contact throughout the bundle, the heat transfer to ambient atmosphere was not hindered. The cathodic end of these capillaries was placed into a cross-flow nebulizer and CEICP-MS experiments were performed. Experimental data showed that the detection limits could easily be improved by a factor of N, where N was the number of loose capillaries in a bundle.
3. Application 3.1. Sample injection Perhaps the most critical stage in CE-based analysis is the sample introduction step. In CE the sample is introduced into the capillary either by electrokinetic injection or by pressure injection Žhydrodynamic.. The experimental approach for electrokinetic injection is depicted in Fig. 6, where a sample is placed at one end of the capillary and it is subjected to a short duration electrical potential. The analytes migrate into the capillary where they can be separated. Because the injection relies on electrical potential, the analytes with higher mobilities are preferentially concentrated on the column. Subsequently, measured concentration of species with vastly varying mobilities is not representative of their indigenous concentrations. Pressure injection does not discriminate against solution composition and it can be achieved in three ways. The procedure for a typical pressure injection is shown in Fig. 7. This type of injection is accomplished either by raising the sample reservoir to a height above the receiving buffer reservoir, by pressurizing the sample reservoir, or
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Fig. 6. Schematic representation of electrokinetic injection technique.
by evacuating the receiving reservoir. In all cases the result is similar, a fixed plug of the sample is introduced into the column and the injection volume can be controlled by duration of the injection period. Interestingly, the negative pressure at the inlet of the nebulizer can be advantageously used for sample introduction in CE-ICP-MS. This suction can be used to force conditioning solutions and cleaning buffers through the capillary, which greatly enhances the flexibility of a CE-
Fig. 7. Schematic representation for different pressure injection techniques.
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ICP-MS system. Furthermore, using predetermined pressure changes with a specific duration we can inject precise amounts of a sample solution onto the CE column. 3.2. Modes of operation CE is family of separation techniques and each technique can be optimized to specifically deal with a particular class of analytes or sample types. Capillary zone electrophoresis is the most frequently used mode of separation where an unmodified fused silica capillary is used to separate charged analytes. The separation mechanism is strictly based on charge-to-mass ratio and this mode of CE is best used for separation of anions and cations. A schematic representation of this mode is shown in Fig. 8. The capillary zone separation of clam MT is illustrated in Fig. 9. The ICP-MS migration profile for clam MT is not identical to those obtained from rabbit MT w22x. Richards and Beattie w29x have shown the variations in MTs as it relates to different organisms and as such different CE-ICP-MS profiles are expected for different animals. As seen for clam metalloproteins, the sensitivity of detection for some elements may not be ideal. One approach to improve the overall detection limit is to preconcentrate the analyte on-column prior to the separation. This can be done using the stacking technique where a 10᎐100-fold sample preconcentration can be obtained. Stack-
Fig. 8. Schematic representation of separation mechanisms for capillary zone electrophoresis.
Fig. 9. CE-ICP-MS electropherogram of clam metallothionein extracted with de-ionized water. The separation was achieved on a 90-cm column at 30 kV using a 50-mM Tris buffer at pH 9.2.
ing is accomplished when the sample matrix has an ionic strength of one or two orders of magnitude lower than the ionic strength of the run buffer. When the sample is injected onto the column and a potential is applied, all charged analytes will rapidly migrate toward the boundary region between the two solutions. This will focus the analyte into a very tight band, which is preserved throughout the separation procedure. The concept of sample stacking is illustrated in Fig. 10. Separation of negative ions in a fused silica capillary is somewhat cumbersome. Although the overall bulk flow Žat higher pH values. toward the detector will eventually force the negative ions into the detector’s path, the experimental time frame is unacceptably long. The easiest way to remedy this problem is to reverse the direction of the electroosmotic flow so that the bulk flow is toward the anode. When reversing the polarity of the imposed potential difference along with flow reversing procedure, the anions can be easily analyzed. To reverse the electroosmotic flow, we have to alter the surface charge characteristics of the fused silica from normally negative to a predominantly positively charged surface. The surface charge can be reversed with cationic surfactants Že.g. dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and cetyltrimethylammonium bromide.. The surfactant forms a double layer near the capillary wall and changes the charge characteristics of the
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Fig. 10. Stacking procedure for capillary electrophoresis. The ionic strength of the sample must be at least a factor of 10 lower than the ionic strength of the run buffer.
capillary surface, hence reversing the direction of the electroosmotic flow. This is shown schematically in Fig. 11. It is important to note that the concentration of surfactant used for reversing the direction of the electroosmotic flow must be maintained below the critical micelle concentrations ŽCMC.. If the concentration of surfactant exceeds CMC then micelles are formed preferentially and a uniform capillary wall coating cannot be attained. The separation of negatively charged arsenic and selenium species is demonstrated in Fig. 12, using ICP-MS detection. Another mode of separation commonly used in CE is the micellar electrokinetic chromatography. In this technique the separation is based on the interaction of analytes with the micelles formed within the separation capillary. Hence, the separation is based on partitioning into and out of the micelles as well as charge-to-mass ratio of ana-
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Fig. 11. Reversing the direction of the electroosmotic flow by coating the fused silica capillary with cationic surfactants at concentrations below the CMC.
lytes. Typically, sodium dodecyl sulfate is used as the surfactant of choice because of it low CMC requirement Ž8.1 mM.. Recalling that higher ionic strength increases the solution conductivity, a sur-
Fig. 12. CE-ICP-MS electropherogram of arsenic and selenium species. The separation was achieved on a 90-cm column at y20 kV using a 20-mM chromate electrolyte at pH 12.0 with 1 mM TTAB Žto reverse the direction of the electroosmotic flow..
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Fig. 13. Micellar electrokinetic chromatography using anionic surfactants at concentrations beyond the CMC.
factant with low CMC can enhance the separation efficiency and the resolution without causing an increase in column temperature due to Ohmic heating. A schematic representation of the micellar electrokinetic chromatography is shown in Fig. 13, and an actual separation of a transferrin protein is shown in Fig. 14. 3.3. Potential problems Perhaps the most important issue in chemical speciation is the concept of measurement traceability as described by Quevauviller w30x. Traceability is a chain of verifiable calibration events that connects the measurement process to the
Fig. 14. Micellar electrokinetic chromatography of transferrin and aluminum mixture. The separation was achieved on a 90-cm column at 30 kV using a 25-mM borate buffer at pH 9.3 with 50 mM SDS Žto generate the micelles..
fundamental units. For complete traceability in elemental speciation one must also verify that the chemical species have not been altered as a result of the analysis process. Publications by Quevauviller w30x, Stewart and Horlick w31x and Donat et al. w32x have discussed the errors caused by tandem use of separation techniques with element specific detectors and the changes in species concentration as a function of kinetic and thermodynamic stability of analytes. When it comes to the analysis of real samples, one has to seriously evaluate the speciation methods presented in the new literature. These techniques are generally tested with stable chemical species within the ideal instrumental parameters, for a given class of compounds Žin a unique matrix.. The analytical results for unknown samples may become questionable if the analyte stability or the sample matrix is altered for a given published technique. Quevauviller w30x has documented the limitations of conventional chromatographic separation when used with element specific detection for speciation applications. The lack of stationary phases in CE-based techniques allows for better representation of labile molecules because of the absence of analyte-stationary phase interactions. Nonetheless, real samples have to be pretreated in most cases to improve sensitivity and separation efficiency. As a result of this pretreatment the species distribution may be influenced within a sample. The following section accentuates some of the factors that can contribute analytical ambiguity in chemical speciation. Additional challenges with regard to the specific sample types and analysis with CE-ICP-MS are highlighted in a manuscript by Olesik et al. w33x. 3.3.1. Interface material Depending on the analyte of interest and the specific buffer system used for the separation procedure, the CE interface material can significantly influence the distribution of species. In some instances new species may be added to the sample because of the interface material or some species may be selectively removed from the sample by the interface. If the interface is predominantly made from noble metals, proteins with
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larger content of cysteine may be selectively adsorbed on to the interface material. In addition, histidine-rich polypeptides are attracted toward nickel, cobalt and Zn metal surfaces. Some separation schemes may require the use of organic modifiers in an aqueous solution or a miscible organicrwater solvent system. In these cases, additional species may be released from the interface if polymeric materials are the main constituent components. Additional breakdown and corrosion takes place at the interface when using buffers with excessively low or high pH values. The surfaces of the metallic interfaces are easily activated at extreme pHs and it could play a dynamic role in the separation process. 3.3.2. Influence of pH In addition to the corrosive effects on the interface, pH plays an important role in the chemical distribution of many species. While raising the pH of a biological sample can greatly simplify the separation, the pertinent information on free metal concentration may be lost due to hydroxideformation Ži.e. formation of precipitates.. The pH contributions are significantly more consequential when the analytes in a sample can interconvert into one another as a result of variations in proton or hydroxide ion concentrations. The equilibrium driven distribution of EDTA᎐metal or any metal᎐biomolecule complex is ultimately defined by the final pH of the system. 3.3.3. Injection parameters In capillary electrophoresis the injection technique and its duration are critical parameters, which may result in a bias in the analytical results. As with any chromatographic technique, longer injection periods will lead to column overloading and, ultimately, poor resolution. This problem is shown in Fig. 15 for both electrokinetic and hydrodynamic injections. In these experiment rabbit MT was injected and separated in a Tris buffer. Different duration of hydrodynamic Žtop inset. and electrokinetic Žbottom inset. injections show slightly different effects. For the hydrodynamic sampling, MT samples were injected using nebulizer gas flow rate of 1.0 lrmin for the specified amount of time Žusing a cross-flow neb-
Fig. 15. Dependence of separation quality in CE-ICP-MS electropherograms for rabbit metallothionein as a function of injection duration for hydrodynamic injection Žtop. and electrokinetic injection Žbottom..
ulizer.. For the electrokinetic injection the sample was subjected to an electrical potential for a short period. The CE run was initiated with a buffer sheath flow rate of 0.375 mlrmin and a nebulizer gas flow rate of 0.7 lrmin Žseparation voltage of 25 kV in 50 mM Tris buffer.. For the hydrodynamic injection, we can see that at lower sample injection duration a better resolution is obtained for different MT isoforms. The column overloading becomes apparent at longer injection duration as evident by the poor resolution. While electrokinetic injection does not precipitously exacerbate column overloading, it does introduce a significant analysis bias toward the analytes with higher mobilities. 3.3.4. Aging The aging process starts when the sample is removed from its original environment. Kinetic
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and thermodynamic factors will begin to influence the analyte distribution as soon as the sample is removed from its native environment. This aging process is most influential in systems where the ambient parameters play an important role in maintaining the balance between different chemical species. This is typically true of biological and environmental samples. For example, MTs have a tendency to polymerize under unfavorable conditions. Subsequently a freshly prepared sample and an aged sample will most likely have different electrophoretic profiles, as demonstrated in paper by Majidi and Miller-Ihli w23x. This article also illustrated the time-dependant Co ion intensity electropherogram from coenzyme-B12, which was rapidly oxidized by the ambient air. 3.3.5. Separation mode Choosing the right separation mode for an unknown sample is a time-consuming process. When tandem techniques are used Že.g. CE-ICPMS. the choice of a specific separation mode becomes more stringent. For example, electrospray ionization technique is generally incompatible with micellar electrochromatography or gelfilled capillary electrophoresis. The choice of solvent system does not significantly influence the detection in ICP-MS, however, it may alter the separation characteristics altogether. For a typical ICP-MS operation, a solution with a few percent nitric acid is the matrix of choice for almost all analytes. Unfortunately, this concept has somewhat propagated into the CE-ICP-MS separation arena. Many investigators, using the CE with ICP-MS, use a dilute solution of nitric acid as the make-up flow solution through the interface. This choice of bulk solution for the make-up flow completely alters the intended mode of separation to isoelectric focusing capillary electrophoresis. The general schematic for isoelectric focusing is shown in Fig. 16. When two different solutions are used at the two ends of the capillary, upon applied potential a pH gradient will be established within the column. The analytes will migrate into the column and stop at a particular pH zone that is equivalent to their isoelectric point. Because most ICP-based species detectors operate at a slightly negative pressure the analyte
Fig. 16. Schematic representation of the separation mechanism in isoelectric focusing capillary electrophoresis.
will eventually be sucked out of the column. Nonetheless, if the intended separation mode was free zone separation or micellar electrokinetic chromatography the user unintentionally will end up using isoelectric focusing technique. 3.4. Speciation in real samples The number of publications on new or improved interfaces is far more voluminous than the application oriented papers for CE-ICP-MS. As this technique matures one can expect for this trend to change in favor of application papers. The most popular applications involve determination of metals or metalloids with an abundance of varying species. Once again, the most frequent applications tend to center around biological and environmental samples. This is where CE-ICP-MS truly outperforms most other techniques. In one case, Michalke and Schramel w34x used CE-ICPMS to evaluate different forms of Se in human milk and serum. Using the isoelectric focusing technique they were able to resolve a high number of selenium species in human serum. The analytical signal, however, was very close to the instrumental noise level. The human milk, on the other hand, provided clear and highly resolved peaks. In a second publication, the same authors demonstrate another example for biological speciation. Working with human serum, Michalke
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and Schramel w35x measured the concentration for different iodine species before and after a thyroid gland operation. The information gained from CE-ICP-MS clearly delineated the role of different hormones in production of various iodine species. The soil extracts and leachates seem to be the second most popular matrix for chemical speciation at this time. Lustig et al. w36x used CE-ICPMS for elemental speciation of Pt compounds in aqueous extracts of clay-like humic soils. In this case, the separation qualities were poor in general, but the authors could distinguish different forms of platinum chloride species. However, Van Holderbeke et al. w37x was more successful in speciation of six different arsenic compounds in soil using CE-ICP-MS technique. In this study the peak shapes were well defined and the availability of standard compounds made the identification of eluted species more conclusive. Recently in a collaborative effort, we have begun to evaluate the level of metal contamination in plants grown in contaminated soils. The plants are homogenized and the biopolymer content, specifically phytochelatens, is extracted with various procedures. Using stacking techniques as a preconcentration scheme, we are able to detect specific metals attached to different biopolymers in specific plants. Fig. 17 is a representative CEICP-MS profile for these species. From the ob-
Fig. 17. CE-ICP-MS electropherogram of biopolymer extracts from P. amurensis. The separation was achieved on a 105-cm column at 29 kV using a 50-mM Tris buffer.
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tained data it is abundantly clear that metals such cadmium and zinc are strongly associated with different types of biopolymers. These studies may someday lead to the use of specific organisms and unique chemical species within these organisms as biomarkers to gauge the health of the environment. References w1x D.R. Baker, Capillary Electrophoresis, John Wiley & Sons Inc, New York, 1995. w2x R.S. Houk, V.A. Fassel, G.D. Flesch, H.J. Svec, A.L. Gray, C.E. Taylor, Anal. Chem. 52 Ž1980. 2283. w3x A. Montaser ŽEd.., Inductively Coupled Plasma Mass Spectrometry, Wiley-VCH, New York, 1998. w4x B.P. Jackson, W.P. Miller, J. Anal. Atom. Spectrom. 13 Ž1998. 1107. w5x M.M. Bayon, M.G. Camblor, J. Anal. Atom. Spectrom. 14 Ž1999. 1317. w6x A. Spiripinyanond, R.M. Barnes, J. Anal. Atom. Spectrom. 14 Ž1999. 1527. w7x R. Lobinski, Appl. Spectrosc. 51 Ž1997. 260A. w8x K. Sutton, R.M.C. Sutton, J.A. Caruso, J. Chromatogr. A 789 Ž1997. 85. w9x F. Vanhaecke, L. Moens, Fresenius J. Anal. Chem. 364 Ž1999. 440. w10x R.S. Houk, Spectrochim. Acta Part B 53 Ž1998. 267. w11x R. Lobinski, H. Chassaigne, J. Szpunar, Talanta 26 Ž1998. 271. w12x B. Michalke, O. Schramel, P. Schramel, Fresenius J. Anal. Chem. 363 Ž1999. 456. w13x J.W. Olesik, J.A. Kinzer, S.V. Olesik, Anal. Chem. 67 Ž1995. 1. w14x J.W. Olesik, J.C. Fister, Spectrochim. Acta Part B 46 Ž1991. 851. w15x J.W. Olesik, S.E. Hobbs, Anal. Chem. 66 Ž1994. 3371. w16x R.M. Barnes, Fresenius J. Anal. Chem. 361 Ž1998. 246. w17x P.W. Kirlew, M.T.M. Castillano, J.A. Caruso, Spectrochim. Acta Part B 53 Ž1998. 221. w18x D. Schaumloffel, A. Prange, Fresenius J. Anal. Chem. 364 Ž1999. 452. w19x Q. Lu, R.M. Barnes, Microchem. J. 54 Ž1996. 129. w20x A. Tangen, W. Lund, B. Josefesson, H. Borg, J. Chromatogr. A 826 Ž1998. 87. w21x K. Taylor, B.L. Sharp, D.J. Lewis, H.M. Crews, J. Anal. Atom. Spectrom. 13 Ž1998. 1095. w22x V. Majidi, N.J. Miller-Ihli, Analyst 123 Ž1998. 803. w23x V. Majidi, N.J. Miller-Ihli, Analyst 123 Ž1998. 809. w24x J.A. Kinzer, J.W. Olesik, S.V. Olesik, Anal. Chem. 68 Ž1996. 3250. w25x Y.Y. Chan, W.T. Chan, J. Chromatogr. A 853 Ž1999. 141. w26x E. Mei, H. Ichihashi, W. Gu, S.I. Yamasaki, Anal. Chem. 69 Ž1997. 2187.
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w27x X.D. Tian, Z.X. Zhuang, B. Chen, X.R. Wang, Analyst 123 Ž1998. 899. w28x V. Majidi, J. Qvarnstrom, Q. Tu, W. Frech, Y. Thomassen, J. Anal. At. Spectrom. 14 Ž1999. 1933. w29x M.P. Richards, J. Beattie, J. Chromatogr. A 648 Ž1993. 459. w30x P. Quevauviller, J. Anal. At. Spectrom. 11 Ž1996. 1225. w31x I.I. Stewart, G. Horlick, J. Anal. At. Spectrom. 11 Ž1996. 1203. w32x J. Donat, K.A. Lao, K.W. Bruland, Anal. Chim. Acta. 284 Ž1994. 547.
w33x J.W. Olesik, J.A. Kinzer, E.J. Grunwald, K.K. Thaxton, S.V. Olesik, Spectrochim. Acta Part B 53 Ž1998. 239. w34x B. Michalke, P. Schramel, J. Chromatogr. A 807 Ž1998. 71. w35x B. Michalke, P. Schramel, Electrophoresis 20 Ž1999. 2547. w36x S. Lustig, B. Michalke, W. Beck, P. Schramel, Fresenius J. Anal. Chem. 360 Ž1998. 18. w37x M. Van Holderbeke, Y. Zhao, F. Vanhaecke, L. Moens, R. Dams, P. Sandra, J. Anal. Atom. Spectrom. 14 Ž1999. 229.
Capillary electrophoresis inductively coupled plasma mass spectrometry Vahid MajidiU Analytical Chemistry Sciences, MS K484, Chemical Science and Technology Di¨ ision, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
Abstract Chemical speciation Žextraction of elemental information and identification of molecular environment for an analyte in a complex sample. has been a long sought after goal for analytical chemists. Recently, because of successful developments in more sensitive element-specific detectors and gentle separation schemes, which preserve the true chemical information in a real sample, routine speciation experiments are becoming a common occurrence in the scientific literature. For many reasons, the combination of capillary electrophoresis Žfor separation of different chemical species. with inductively coupled plasma mass spectrometry Žfor element and isotope specific detection. has emerged as the method of choice for these analyses. In this article the basic principles of capillary electrophoresis inductively coupled plasma mass spectrometry are discussed. Design consideration for instrument interface, anticipated difficulties with speciation experiments and applications for specific matrices and analytes are also presented in this article. 䊚 2000 Published by Elsevier Science B.V. Keywords: Capillary electrophoresis; Inductively coupled plasma mass spectrometry
1. Introduction Continuing technological development in separation science and spectrochemical detection has ultimately resulted in evolutionary improvements in hyphenated and tandem techniques. Combination of two or more analytical techniques always
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Tel.: q1-505-667-0040. E-mail address: [email protected] ŽV. Majidi..
provides a higher data density than either technique individually. This has been the prevalent trend in instrument development research during the past decade. In the 1980s, the spectroscopy and the separation science communities were witness to a parallel development and maturation of two powerful instrumental techniques: capillary electrophoresis ŽCE. and inductively coupled plasma mass spectrometry ŽICP-MS.. Capillary electrophoresis became commercially available in the latter part of
0026-265Xr00r$ - see front matter 䊚 2000 Published by Elsevier Science B.V. PII: S 0 0 2 6 - 2 6 5 X Ž 0 0 . 0 0 0 6 2 - X
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the 1980s and rapidly emerged as a routine tool for many clinical and pharmaceutical applications. In reality, capillary electrophoresis is a family of separation techniques based on electrophoretic migration of analytes in a capillary w1x. Among the positive attributes associated with CE, perhaps the most important advantage with regard to chemical speciation applications is the lack of a stationary phase in the separation column. When dealing with kinetically or thermodynamically labile molecules, the analyte interactions with a stationary phase in conventional chromatographic separation techniques may be detrimental to the analysis. That is, the nature of the analytes introduced onto the separation column may be altered because of their interactions with the stationary phase. In CE methods, the electrophoretic forces drive the separation and as such there is no need for a stationary phase to impose specific chemical interactions. In one case, electrochromatography, a packed capillary is used to allow for distinction between similar compounds. Nevertheless, capillary electrochromatography does not really belong to the CE family and the electrical potential imposed across the column is merely a ‘pump’ to generate an electroosmotic flow through the column. Another significant analytical development during the 1980s was the invention and commercialization of inductively coupled plasma mass spectrometry, first described by Houk et al. w2x. This hybrid instrument exploits the energetic plasma in an ICP to completely destroy the sample matrix and efficiently ionize the constituent elements. When an ICP source is combined with mass spectrometric detection, one of the most sensitive element-specific detectors is obtained Žbecause the majority of the elements passing through the plasma are nearly completely ionized.. ICP-MS can provide quantitative elemental and isotopic information with a dynamic range exceeding five orders of magnitude and instrumental limits of detection better than 1 ngrl for most elements w3x. Although this instrument can provide nearly simultaneous multi-elemental analysis, because of the energetic nature of inductive plasma, the speciation information is not preserved.
Extraction of elemental speciation information from small samples or dilute analytes is best achieved through the on-line combination of ICPMS with an instrument capable of separating analytes based on their chemical w4,5x or physical properties w6x. The readers interested in coupled techniques for chemical speciation will find the comprehensive review articles by Lobinski w7x, Sutton et al. w8x and Vanhaecke and Moens w9x most useful. From the list of potential coupled techniques, the combination of CE with ICP-MS seems to provide a set of performance characteristics unmatched by any other technique. Perhaps, the second most versatile detection approach for CE is the electrospray technique. Unfortunately, not every bufferranalyte combination is amenable to analysis by the electrospray technique, the detection limits are not as good as ICP-MS and the linear dynamic range is extremely limited. These two approaches should not be considered as competitive but rather complementary techniques. Houk w10x, Lobinski et al. w11x and Michalke et al. w12x have reported comparison of these two techniques for various types of analysis. It is not surprising that the potential utility of CE in tandem with ICP-MS was recognized relatively early in the speciation community. However, perceived difficulties in designing a functional interface for coupling of CE with ICP-MS discouraged most researchers from this venture. The operational parameters for CE and ICP-MS experiments were individually well established. However, these parameters are not necessarily compatible between the two techniques. For example, the flow rate of run buffer in a CE column can be as little as a few nlrmin, while the conventional ICP-MS sample introduction requires a flow rate of approximately 1 mlrmin. The requirements for electrical continuity along with six orders of magnitude difference in flow rates between the two systems were the main reason for the postponement of an interface development to the mid-1990s. It is fitting that Olesik et al. w13x reported the first functional interface for CE-ICP-MS. Olesik w14,15x had previously worked specifically on evaluating the sample uptake, aerosol formation, aerosol transport, and the atomization mechanisms in both ICP and ICP-MS.
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Armed with this knowledge, Olesik understood that the ICP-MS could operate effectively with smaller sample flow rates and that at these small flow rates Žlrmin. the aerosol formation and transport are very efficient. Using this basic concept and a small make-up flow, a successful CEICP-MS coupling was achieved. Today, almost every combination of nebulizers and spray chambers has been exploited for CE-ICP-MS applications. In a brief review, Barnes w16x describes a general overview of CE-ICP-MS technique, instrumental requirement and the potential for microfabrication. Interested readers may also find the manuscript on use of ultrasonic nebulizers for CE-ICP-MS applications, by Kirlew et al. w17x, relevant to the content of this article.
2. Instrumentation It is not the intention of this article to fully cover the instrumental details associated with CE or ICP-MS. Several excellent books with specific focus on each specific technique are available on these subjects w1,3x. Our goal is to present a brief overview of these techniques so that the difficulties associated with using them in a tandem arrangement will be apparent and the solutions used to deal with these issues can be better appreciated. 2.1. CE Capillary electrophoresis is a simple and powerful separation technique. The separation is often achieved in a fused silica capillary with an internal diameter of 25᎐100 m. The instrument is completely operational when the capillary is filled with a buffer and both ends of the column are submerged in two different buffer reservoirs equipped with electrical connections. Platinum electrodes are used to establish a potential difference between the two ends of the capillary. A potential difference of up to 30 kV is applied to the 1-m-long capillary, which results in electric fields of up to 300 Vrcm. Typically, the maximum potential is selected so that the run currents are maintained below 100 A. The general schematic
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Fig. 1. A block diagram for conventional capillary electrophoresis instrument with an UV detector.
arrangement of a CE instrument is shown in Fig. 1. In CE the absolute potential difference between the two end of the capillary is not, by itself, a critical parameter. However, the electric field imposed on the capillary Žpotential difference divided by the length of the capillary. ultimately determines the resolution and separation efficiency. Parameters that profoundly influence the CE separation include the types of ions present, the internal diameter of the capillary, buffer ionic strength and pH. In most instances, these factors work in concert to effect the separation characteristics. For example, by increasing the ionic strength of the buffer, better resolution is often expected. As the ionic strength of the solution is increased the temperature of the run buffer becomes higher because of increased current for a given potential difference Žresistive heating.. This in turn leads to better thermal diffusion of analytes resulting in an exacerbated resolution. Use of smaller diameter capillaries will allow for reduced run currents and a more efficient cooling of the capillary. These interdependencies are not a mere linear function of one another, and subsequently, optimized run conditions have to be established for a unique combination of buffer, capillary and applied electric field. Fortunately, most of these combined parameters have been evaluated and presented in the literature. If a unique set of parameters is being used, as expected for application of CE in tandem techniques, an Ohm-law plot should be constructed to avoid some unacceptable experimental conditions. The Ohm-law plot simply shows the depen-
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dency of the observed run current as a function of applied potential across the capillary. Ideally, a linear relationship between the applied potential and the measured current should be obtained Ž V s IR, where V is the applied potential, I is the measured current and R is the resistance of the buffer solution within the column.. At higher potentials, however, this linear response may not be observed as depicted in Fig. 2. The best run condition is commonly selected at the highest possible potential within the linear region of the Ohm-law plot. In a gross oversimplification we can state that the best separation conditions are obtained in capillaries with very small diameters, at high ionic strengths and large imposed electric fields.
Fig. 2. A plot of measured and expected current in a CE column as a function of applied potential.
2.2. ICP-MS In contrast to CE, ICP-MS is a highly complex instrument that relies on two decades of technology development in RF plasmas and ion optics for mass spectrometric detection. Today’s instruments are highly automated based on a series of predetermined experimental parameters programmed by manufacturers. To some extent, this high level of automation is a nuisance for tandem instrumental applications because the optimized parameters for the operation of the ICP-MS in a hyphenated mode may not be similar to the conditions optimized for ICP-MS alone. As we mentioned previously, detailed description of ICP-MS instrumentation can be found elsewhere w3x. Briefly, this instrument consists of a plasma torch ion source and a mass analyzer. The plasma is generated in an Ar environment by a high-power radio-frequency generator Žapprox. 1 kW.. The resulting plasma possesses high excitation and ionization temperature. The species traveling through this plasma are atomized and ionized with a very high efficiency. The ions are extracted from the ICP torch into a mass analyzer through a two-stage, differentially pumped, sampling and skimmer cone assembly. The sampled ions are directed toward the mass analyzer Žoften a quadrupole mass filter. with a series of ion optics assemblies. A schematic diagram of the ICP-MS is shown in Fig. 3.
Fig. 3. A simplified schematic representation of an ICP-MS instrument.
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2.3. Interface The interface can be best represented as a combination of three subsystems: spray chamber, nebulizer and the CE connection hardware. While the manuscripts published on this topic often contain the phrase ‘a new interface’ Že.g. Schaumloffel and Prange w18x. in their corresponding titles, in reality they refer to an alternative combination of CE-connection with different nebulizerrspray chamber arrangement. Nonetheless, Lu and Barnes w19x, Tangen et al. w20x and Taylor et al. w21x have described some innovative combinations of the above three components for CE-ICP-MS instrumentation. The basic CE-connection hardware to any nebulizer is virtually the same in all publications with only minor variations in some instances w22,23x. The schematic of this generalized CE-connection to a generic nebulizer is shown in Fig. 4. As shown in the figure, the separation capillary is inserted through a Peek tee Žmodel P-728. and it is held in place using a model F-300 Peek fitting and a 0.4-mm Vespel ferrule ŽAlltech Associates Inc., Deerfield, IL, USA.. All tees, fittings, PEEK ferrules, and Teflon tubing were purchased from Upchurch Scientific ŽOak Harbor, WA, USA.. The sheath buffer flow is mixed with the CE effluent prior to nebulization and the mixing of the buffer solutions occurs at the tip of the separation capillary Žthe buffer sheath flow is also the electrical connection for the circuit continuity.. Teflon tubing Ž0.76 mm i.d.=1.6 mm o.d.. attached to a peristaltic pump supplies the buffer flow to the interface. The cathodic electrical connection is made through the same make-up buffer port of the interface tee by placing a 0.076 mm o.d. platinum wire between the Teflon tubing and the Peek ferrule. Another 0.76 mm i.d. Teflon tubing is used to guide the buffer sheath flow and the separation capillary into the tip of the nebulizer. The above CE connection can be attached to any nebulizer to facilitate analyte transport into the ICP torch. Some of the most commonly used combination of nebulizers and spray chambers is shown in Fig. 5. These combinations of CErnebulizerrspray chamber are all functional, and their performance mostly depends on the
Fig. 4. The general interface design for capillary electrophoresis.
specific goals for each analysis. In some instances the performance of these interfaces have been handicapped because of the self-aspirating nature of the commercial pneumatic nebulizers. If not regulated, the aspiration in these nebulizers causes a significant laminar flow within the capillary. Kinzer et al. w24x has discussed the consequence of this laminar flow. The aspiration rate of each nebulizer is a function of the Ar gas flow rate and the nebulizer design. The data shows that an increase in the nebulizer gas flow rate will result in faster migration velocities of analytes within the capillary. The negative pressure at the inlet of the nebulizer causes this suction of the analyte in the capillary. Nonetheless, this problem can be easily resolved or minimized with judicious use of makeup buffer flow and nebulizer gas flow rates. In general, when attempting to use the commercial nebulizerrspray systems for CE applications, one has to consider the parameters that influence the separation efficiencies as well
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detection limits. Total dead volumes, composition of buffers and flow compatibilities are also among the parameters that must be optimized for a successful union of CE with ICP-MS system. 2.4. Unique approaches Stability and ease of fabrication of the above simple interface is responsible for its vast popularity. Other unique interfaces have been designed for transport of CE effluent into the ICP torch, but with limited success. One successful design, by Chan and Chan w25x, uses an integrated approach for analyte sampling. In this design, the capillary is made of two segments connected by a low dead-volume tee. This tee provides the electrical continuity for the CE separation as well as being the outflow channel toward the ICP torch.
Another design promoted by Mei et al. w26x completely eliminates the need for a makeup buffer and the electrical connection is made directly through a thin Pt wire inserted into the cathodic end of the capillary. In either of the above cases the control and optimization of separation and analyte introduction parameters is non-trivial. Tian et al. w27x published a clever concept for connection of CE to an ICP torch. Recognizing that hydride generation can significantly improve the detection limits for hydride forming elements, they designed an interface based on a movable reduction bed hydride generation system. The advantages of such system include excellent analyte transport and some analyte pre-speciation based on hydride formation selectivity. Because of the hydride generation step, this system cannot be used with all elements.
Fig. 5. Different nebulizers and spray chamber configurations typically used for CE-ICP-MS.
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Considering that the sample loading in CE is typically less than 100 nl, the major drawback, when using CE for chemical speciation, is the limited amount of sample utilized for the analysis. Recognizing this limitation, Majidi et al. w28x published a unique approach, which takes advantage of parallel separation scheme to realistically improve the analytical sensitivity and detection limits. Several individual capillaries with identical internal diameters and lengths were placed in a loose bundle and because the capillaries were not physically in contact throughout the bundle, the heat transfer to ambient atmosphere was not hindered. The cathodic end of these capillaries was placed into a cross-flow nebulizer and CEICP-MS experiments were performed. Experimental data showed that the detection limits could easily be improved by a factor of N, where N was the number of loose capillaries in a bundle.
3. Application 3.1. Sample injection Perhaps the most critical stage in CE-based analysis is the sample introduction step. In CE the sample is introduced into the capillary either by electrokinetic injection or by pressure injection Žhydrodynamic.. The experimental approach for electrokinetic injection is depicted in Fig. 6, where a sample is placed at one end of the capillary and it is subjected to a short duration electrical potential. The analytes migrate into the capillary where they can be separated. Because the injection relies on electrical potential, the analytes with higher mobilities are preferentially concentrated on the column. Subsequently, measured concentration of species with vastly varying mobilities is not representative of their indigenous concentrations. Pressure injection does not discriminate against solution composition and it can be achieved in three ways. The procedure for a typical pressure injection is shown in Fig. 7. This type of injection is accomplished either by raising the sample reservoir to a height above the receiving buffer reservoir, by pressurizing the sample reservoir, or
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Fig. 6. Schematic representation of electrokinetic injection technique.
by evacuating the receiving reservoir. In all cases the result is similar, a fixed plug of the sample is introduced into the column and the injection volume can be controlled by duration of the injection period. Interestingly, the negative pressure at the inlet of the nebulizer can be advantageously used for sample introduction in CE-ICP-MS. This suction can be used to force conditioning solutions and cleaning buffers through the capillary, which greatly enhances the flexibility of a CE-
Fig. 7. Schematic representation for different pressure injection techniques.
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ICP-MS system. Furthermore, using predetermined pressure changes with a specific duration we can inject precise amounts of a sample solution onto the CE column. 3.2. Modes of operation CE is family of separation techniques and each technique can be optimized to specifically deal with a particular class of analytes or sample types. Capillary zone electrophoresis is the most frequently used mode of separation where an unmodified fused silica capillary is used to separate charged analytes. The separation mechanism is strictly based on charge-to-mass ratio and this mode of CE is best used for separation of anions and cations. A schematic representation of this mode is shown in Fig. 8. The capillary zone separation of clam MT is illustrated in Fig. 9. The ICP-MS migration profile for clam MT is not identical to those obtained from rabbit MT w22x. Richards and Beattie w29x have shown the variations in MTs as it relates to different organisms and as such different CE-ICP-MS profiles are expected for different animals. As seen for clam metalloproteins, the sensitivity of detection for some elements may not be ideal. One approach to improve the overall detection limit is to preconcentrate the analyte on-column prior to the separation. This can be done using the stacking technique where a 10᎐100-fold sample preconcentration can be obtained. Stack-
Fig. 8. Schematic representation of separation mechanisms for capillary zone electrophoresis.
Fig. 9. CE-ICP-MS electropherogram of clam metallothionein extracted with de-ionized water. The separation was achieved on a 90-cm column at 30 kV using a 50-mM Tris buffer at pH 9.2.
ing is accomplished when the sample matrix has an ionic strength of one or two orders of magnitude lower than the ionic strength of the run buffer. When the sample is injected onto the column and a potential is applied, all charged analytes will rapidly migrate toward the boundary region between the two solutions. This will focus the analyte into a very tight band, which is preserved throughout the separation procedure. The concept of sample stacking is illustrated in Fig. 10. Separation of negative ions in a fused silica capillary is somewhat cumbersome. Although the overall bulk flow Žat higher pH values. toward the detector will eventually force the negative ions into the detector’s path, the experimental time frame is unacceptably long. The easiest way to remedy this problem is to reverse the direction of the electroosmotic flow so that the bulk flow is toward the anode. When reversing the polarity of the imposed potential difference along with flow reversing procedure, the anions can be easily analyzed. To reverse the electroosmotic flow, we have to alter the surface charge characteristics of the fused silica from normally negative to a predominantly positively charged surface. The surface charge can be reversed with cationic surfactants Že.g. dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and cetyltrimethylammonium bromide.. The surfactant forms a double layer near the capillary wall and changes the charge characteristics of the
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Fig. 10. Stacking procedure for capillary electrophoresis. The ionic strength of the sample must be at least a factor of 10 lower than the ionic strength of the run buffer.
capillary surface, hence reversing the direction of the electroosmotic flow. This is shown schematically in Fig. 11. It is important to note that the concentration of surfactant used for reversing the direction of the electroosmotic flow must be maintained below the critical micelle concentrations ŽCMC.. If the concentration of surfactant exceeds CMC then micelles are formed preferentially and a uniform capillary wall coating cannot be attained. The separation of negatively charged arsenic and selenium species is demonstrated in Fig. 12, using ICP-MS detection. Another mode of separation commonly used in CE is the micellar electrokinetic chromatography. In this technique the separation is based on the interaction of analytes with the micelles formed within the separation capillary. Hence, the separation is based on partitioning into and out of the micelles as well as charge-to-mass ratio of ana-
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Fig. 11. Reversing the direction of the electroosmotic flow by coating the fused silica capillary with cationic surfactants at concentrations below the CMC.
lytes. Typically, sodium dodecyl sulfate is used as the surfactant of choice because of it low CMC requirement Ž8.1 mM.. Recalling that higher ionic strength increases the solution conductivity, a sur-
Fig. 12. CE-ICP-MS electropherogram of arsenic and selenium species. The separation was achieved on a 90-cm column at y20 kV using a 20-mM chromate electrolyte at pH 12.0 with 1 mM TTAB Žto reverse the direction of the electroosmotic flow..
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Fig. 13. Micellar electrokinetic chromatography using anionic surfactants at concentrations beyond the CMC.
factant with low CMC can enhance the separation efficiency and the resolution without causing an increase in column temperature due to Ohmic heating. A schematic representation of the micellar electrokinetic chromatography is shown in Fig. 13, and an actual separation of a transferrin protein is shown in Fig. 14. 3.3. Potential problems Perhaps the most important issue in chemical speciation is the concept of measurement traceability as described by Quevauviller w30x. Traceability is a chain of verifiable calibration events that connects the measurement process to the
Fig. 14. Micellar electrokinetic chromatography of transferrin and aluminum mixture. The separation was achieved on a 90-cm column at 30 kV using a 25-mM borate buffer at pH 9.3 with 50 mM SDS Žto generate the micelles..
fundamental units. For complete traceability in elemental speciation one must also verify that the chemical species have not been altered as a result of the analysis process. Publications by Quevauviller w30x, Stewart and Horlick w31x and Donat et al. w32x have discussed the errors caused by tandem use of separation techniques with element specific detectors and the changes in species concentration as a function of kinetic and thermodynamic stability of analytes. When it comes to the analysis of real samples, one has to seriously evaluate the speciation methods presented in the new literature. These techniques are generally tested with stable chemical species within the ideal instrumental parameters, for a given class of compounds Žin a unique matrix.. The analytical results for unknown samples may become questionable if the analyte stability or the sample matrix is altered for a given published technique. Quevauviller w30x has documented the limitations of conventional chromatographic separation when used with element specific detection for speciation applications. The lack of stationary phases in CE-based techniques allows for better representation of labile molecules because of the absence of analyte-stationary phase interactions. Nonetheless, real samples have to be pretreated in most cases to improve sensitivity and separation efficiency. As a result of this pretreatment the species distribution may be influenced within a sample. The following section accentuates some of the factors that can contribute analytical ambiguity in chemical speciation. Additional challenges with regard to the specific sample types and analysis with CE-ICP-MS are highlighted in a manuscript by Olesik et al. w33x. 3.3.1. Interface material Depending on the analyte of interest and the specific buffer system used for the separation procedure, the CE interface material can significantly influence the distribution of species. In some instances new species may be added to the sample because of the interface material or some species may be selectively removed from the sample by the interface. If the interface is predominantly made from noble metals, proteins with
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larger content of cysteine may be selectively adsorbed on to the interface material. In addition, histidine-rich polypeptides are attracted toward nickel, cobalt and Zn metal surfaces. Some separation schemes may require the use of organic modifiers in an aqueous solution or a miscible organicrwater solvent system. In these cases, additional species may be released from the interface if polymeric materials are the main constituent components. Additional breakdown and corrosion takes place at the interface when using buffers with excessively low or high pH values. The surfaces of the metallic interfaces are easily activated at extreme pHs and it could play a dynamic role in the separation process. 3.3.2. Influence of pH In addition to the corrosive effects on the interface, pH plays an important role in the chemical distribution of many species. While raising the pH of a biological sample can greatly simplify the separation, the pertinent information on free metal concentration may be lost due to hydroxideformation Ži.e. formation of precipitates.. The pH contributions are significantly more consequential when the analytes in a sample can interconvert into one another as a result of variations in proton or hydroxide ion concentrations. The equilibrium driven distribution of EDTA᎐metal or any metal᎐biomolecule complex is ultimately defined by the final pH of the system. 3.3.3. Injection parameters In capillary electrophoresis the injection technique and its duration are critical parameters, which may result in a bias in the analytical results. As with any chromatographic technique, longer injection periods will lead to column overloading and, ultimately, poor resolution. This problem is shown in Fig. 15 for both electrokinetic and hydrodynamic injections. In these experiment rabbit MT was injected and separated in a Tris buffer. Different duration of hydrodynamic Žtop inset. and electrokinetic Žbottom inset. injections show slightly different effects. For the hydrodynamic sampling, MT samples were injected using nebulizer gas flow rate of 1.0 lrmin for the specified amount of time Žusing a cross-flow neb-
Fig. 15. Dependence of separation quality in CE-ICP-MS electropherograms for rabbit metallothionein as a function of injection duration for hydrodynamic injection Žtop. and electrokinetic injection Žbottom..
ulizer.. For the electrokinetic injection the sample was subjected to an electrical potential for a short period. The CE run was initiated with a buffer sheath flow rate of 0.375 mlrmin and a nebulizer gas flow rate of 0.7 lrmin Žseparation voltage of 25 kV in 50 mM Tris buffer.. For the hydrodynamic injection, we can see that at lower sample injection duration a better resolution is obtained for different MT isoforms. The column overloading becomes apparent at longer injection duration as evident by the poor resolution. While electrokinetic injection does not precipitously exacerbate column overloading, it does introduce a significant analysis bias toward the analytes with higher mobilities. 3.3.4. Aging The aging process starts when the sample is removed from its original environment. Kinetic
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and thermodynamic factors will begin to influence the analyte distribution as soon as the sample is removed from its native environment. This aging process is most influential in systems where the ambient parameters play an important role in maintaining the balance between different chemical species. This is typically true of biological and environmental samples. For example, MTs have a tendency to polymerize under unfavorable conditions. Subsequently a freshly prepared sample and an aged sample will most likely have different electrophoretic profiles, as demonstrated in paper by Majidi and Miller-Ihli w23x. This article also illustrated the time-dependant Co ion intensity electropherogram from coenzyme-B12, which was rapidly oxidized by the ambient air. 3.3.5. Separation mode Choosing the right separation mode for an unknown sample is a time-consuming process. When tandem techniques are used Že.g. CE-ICPMS. the choice of a specific separation mode becomes more stringent. For example, electrospray ionization technique is generally incompatible with micellar electrochromatography or gelfilled capillary electrophoresis. The choice of solvent system does not significantly influence the detection in ICP-MS, however, it may alter the separation characteristics altogether. For a typical ICP-MS operation, a solution with a few percent nitric acid is the matrix of choice for almost all analytes. Unfortunately, this concept has somewhat propagated into the CE-ICP-MS separation arena. Many investigators, using the CE with ICP-MS, use a dilute solution of nitric acid as the make-up flow solution through the interface. This choice of bulk solution for the make-up flow completely alters the intended mode of separation to isoelectric focusing capillary electrophoresis. The general schematic for isoelectric focusing is shown in Fig. 16. When two different solutions are used at the two ends of the capillary, upon applied potential a pH gradient will be established within the column. The analytes will migrate into the column and stop at a particular pH zone that is equivalent to their isoelectric point. Because most ICP-based species detectors operate at a slightly negative pressure the analyte
Fig. 16. Schematic representation of the separation mechanism in isoelectric focusing capillary electrophoresis.
will eventually be sucked out of the column. Nonetheless, if the intended separation mode was free zone separation or micellar electrokinetic chromatography the user unintentionally will end up using isoelectric focusing technique. 3.4. Speciation in real samples The number of publications on new or improved interfaces is far more voluminous than the application oriented papers for CE-ICP-MS. As this technique matures one can expect for this trend to change in favor of application papers. The most popular applications involve determination of metals or metalloids with an abundance of varying species. Once again, the most frequent applications tend to center around biological and environmental samples. This is where CE-ICP-MS truly outperforms most other techniques. In one case, Michalke and Schramel w34x used CE-ICPMS to evaluate different forms of Se in human milk and serum. Using the isoelectric focusing technique they were able to resolve a high number of selenium species in human serum. The analytical signal, however, was very close to the instrumental noise level. The human milk, on the other hand, provided clear and highly resolved peaks. In a second publication, the same authors demonstrate another example for biological speciation. Working with human serum, Michalke
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and Schramel w35x measured the concentration for different iodine species before and after a thyroid gland operation. The information gained from CE-ICP-MS clearly delineated the role of different hormones in production of various iodine species. The soil extracts and leachates seem to be the second most popular matrix for chemical speciation at this time. Lustig et al. w36x used CE-ICPMS for elemental speciation of Pt compounds in aqueous extracts of clay-like humic soils. In this case, the separation qualities were poor in general, but the authors could distinguish different forms of platinum chloride species. However, Van Holderbeke et al. w37x was more successful in speciation of six different arsenic compounds in soil using CE-ICP-MS technique. In this study the peak shapes were well defined and the availability of standard compounds made the identification of eluted species more conclusive. Recently in a collaborative effort, we have begun to evaluate the level of metal contamination in plants grown in contaminated soils. The plants are homogenized and the biopolymer content, specifically phytochelatens, is extracted with various procedures. Using stacking techniques as a preconcentration scheme, we are able to detect specific metals attached to different biopolymers in specific plants. Fig. 17 is a representative CEICP-MS profile for these species. From the ob-
Fig. 17. CE-ICP-MS electropherogram of biopolymer extracts from P. amurensis. The separation was achieved on a 105-cm column at 29 kV using a 50-mM Tris buffer.
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tained data it is abundantly clear that metals such cadmium and zinc are strongly associated with different types of biopolymers. These studies may someday lead to the use of specific organisms and unique chemical species within these organisms as biomarkers to gauge the health of the environment. References w1x D.R. Baker, Capillary Electrophoresis, John Wiley & Sons Inc, New York, 1995. w2x R.S. Houk, V.A. Fassel, G.D. Flesch, H.J. Svec, A.L. Gray, C.E. Taylor, Anal. Chem. 52 Ž1980. 2283. w3x A. Montaser ŽEd.., Inductively Coupled Plasma Mass Spectrometry, Wiley-VCH, New York, 1998. w4x B.P. Jackson, W.P. Miller, J. Anal. Atom. Spectrom. 13 Ž1998. 1107. w5x M.M. Bayon, M.G. Camblor, J. Anal. Atom. Spectrom. 14 Ž1999. 1317. w6x A. Spiripinyanond, R.M. Barnes, J. Anal. Atom. Spectrom. 14 Ž1999. 1527. w7x R. Lobinski, Appl. Spectrosc. 51 Ž1997. 260A. w8x K. Sutton, R.M.C. Sutton, J.A. Caruso, J. Chromatogr. A 789 Ž1997. 85. w9x F. Vanhaecke, L. Moens, Fresenius J. Anal. Chem. 364 Ž1999. 440. w10x R.S. Houk, Spectrochim. Acta Part B 53 Ž1998. 267. w11x R. Lobinski, H. Chassaigne, J. Szpunar, Talanta 26 Ž1998. 271. w12x B. Michalke, O. Schramel, P. Schramel, Fresenius J. Anal. Chem. 363 Ž1999. 456. w13x J.W. Olesik, J.A. Kinzer, S.V. Olesik, Anal. Chem. 67 Ž1995. 1. w14x J.W. Olesik, J.C. Fister, Spectrochim. Acta Part B 46 Ž1991. 851. w15x J.W. Olesik, S.E. Hobbs, Anal. Chem. 66 Ž1994. 3371. w16x R.M. Barnes, Fresenius J. Anal. Chem. 361 Ž1998. 246. w17x P.W. Kirlew, M.T.M. Castillano, J.A. Caruso, Spectrochim. Acta Part B 53 Ž1998. 221. w18x D. Schaumloffel, A. Prange, Fresenius J. Anal. Chem. 364 Ž1999. 452. w19x Q. Lu, R.M. Barnes, Microchem. J. 54 Ž1996. 129. w20x A. Tangen, W. Lund, B. Josefesson, H. Borg, J. Chromatogr. A 826 Ž1998. 87. w21x K. Taylor, B.L. Sharp, D.J. Lewis, H.M. Crews, J. Anal. Atom. Spectrom. 13 Ž1998. 1095. w22x V. Majidi, N.J. Miller-Ihli, Analyst 123 Ž1998. 803. w23x V. Majidi, N.J. Miller-Ihli, Analyst 123 Ž1998. 809. w24x J.A. Kinzer, J.W. Olesik, S.V. Olesik, Anal. Chem. 68 Ž1996. 3250. w25x Y.Y. Chan, W.T. Chan, J. Chromatogr. A 853 Ž1999. 141. w26x E. Mei, H. Ichihashi, W. Gu, S.I. Yamasaki, Anal. Chem. 69 Ž1997. 2187.
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w27x X.D. Tian, Z.X. Zhuang, B. Chen, X.R. Wang, Analyst 123 Ž1998. 899. w28x V. Majidi, J. Qvarnstrom, Q. Tu, W. Frech, Y. Thomassen, J. Anal. At. Spectrom. 14 Ž1999. 1933. w29x M.P. Richards, J. Beattie, J. Chromatogr. A 648 Ž1993. 459. w30x P. Quevauviller, J. Anal. At. Spectrom. 11 Ž1996. 1225. w31x I.I. Stewart, G. Horlick, J. Anal. At. Spectrom. 11 Ž1996. 1203. w32x J. Donat, K.A. Lao, K.W. Bruland, Anal. Chim. Acta. 284 Ž1994. 547.
w33x J.W. Olesik, J.A. Kinzer, E.J. Grunwald, K.K. Thaxton, S.V. Olesik, Spectrochim. Acta Part B 53 Ž1998. 239. w34x B. Michalke, P. Schramel, J. Chromatogr. A 807 Ž1998. 71. w35x B. Michalke, P. Schramel, Electrophoresis 20 Ž1999. 2547. w36x S. Lustig, B. Michalke, W. Beck, P. Schramel, Fresenius J. Anal. Chem. 360 Ž1998. 18. w37x M. Van Holderbeke, Y. Zhao, F. Vanhaecke, L. Moens, R. Dams, P. Sandra, J. Anal. Atom. Spectrom. 14 Ž1999. 229.