Chapter 6 Capillary electrophoresis for elemental speciation studies

Chapter 6

Capillary electrophoresis for elemental speciation studies John W. Olesik

6.1 INTRODUCTION Capillary electrophoresis (CE) can provide high resolution, high efficiency and rapid analysis times for a variety of species including cations, anions, small metal ions, metal-organic ligand complexes, organometallic molecules and biomacromolecules [1]. The principles of capillary electrophoresis were described in the early 1980s [2]. While CE has become most popular for separation of macromolecules, CE has been used for the analysis of metal ions since its early development [3,4]. Capillary ion electrophoresis has been used for low molecular weight anions and cations, mainly with indirect UV absorption detection [5]. Dabek-Zlotorzynska et al. [6] recently reviewed the use of CE for metal speciation, which has been growing rapidly during the last five years. An entire issue of the Journal of Chromatography A was dedicated to the electrophoresis of inorganic species with several articles about elemental speciation using CE [7]. The growth of CE for elemental speciation will be accelerated by the use of element selective and molecule selective detectors with lower detection limits than nonspecific UV absorption based detection. The current status of the CE with ICP-MS element selective detection, has been recently reviewed by Barnes [8]. CE has some unique characteristics that make it particularly attractive for elemental speciation although gas chromatography and liquid chromatography are more widely used. A diverse range of sample types can be analyzed with high efficiency. Only small (less than pl) sample sizes are required. Capillary electrophoresis, Comprehensive Analytical Chemistry, Vol. XXXIII J.A. Caruso, K.L. Sutton and K.L. Ackley (Eds.) ( 2000 Elsevier Science B.V. All rights reserved

151

sometimes called free-solution CE or capillary zone electrophoresis, separates analytes on the basis of their mobility in an electric field, rather than a chemical interaction and partitioning between a stationary phase and a mobile phase. The mobility of an analyte depends on its charge, size and shape. Therefore, the CE migration order for many species of interest can be predicted well. This becomes particularly advantageous when CE is coupled with an element selective detector so that only different forms of a particular element of interest must be separated from each other rather than needing to separate species containing different elements from each other. Tradeoffs between resolution, efficiency and analysis times are easily controlled by the applied electric field gradient, the time analytes spend in the capillary under the influence of the electric field gradient and the laminar flow rate. Because the CE separation is ideally based on the movement of ions under the influence of an electric field rather than chemical interactions, some, including this author, have suggested that CE may disturb the existing distribution of species less severely than chromatographic methods [6,9,10]. However, this will depend critically on avoiding species-wall interactions and on choosing the appropriate electrolyte/buffer solutions. One of the biggest problems in using CE for elemental speciation is that the sample size can be so small (10-100 nl, typically). As a result, detectors that have a sensitivity that depends either on path length (such as UV visible absorption) or the amount (mass) of analyte that enters the detector per second (such as plasma spectroscopy and mass spectrometry) may suffer from degraded concentration based detection limits. Two other problems can deleteriously affect CE for elemental speciation. Undesirable capillary wall-sample interactions (due to chemical interactions as well as trapping in the electrical double layer) can affect analyte migration times and migration time reproducibility. Analyte interactions with the capillary wall or the static solution layer near the wall can also result in significant sample trapping in the capillary. As a result, capillary pretreatment procedures and capillary coatings can be keys to successful CE separations. Electrophoretic resolution is degraded if the conductivity of the sample is not significantly less than that of the electrolyte/buffer in the capillary. Joule heating limits the maximum conductivity of CE electrolytes/buffers that can be used. In this chapter, the fundamental parameters that affect capillary electrophoresis separations will be briefly examined along with the 152

instrumentation for CE. Some examples of types of analytes that are amenable to CE separation will be illustrated. Modes of injecting the sample into the capillary will be considered. On-capillary and postcapillary detectors will be described. A particular emphasis will be placed on interface considerations for post-capillary, element and molecule selective detection. Potential problems resulting from capillary wall-sample interactions will be evaluated. The applications of CE for elemental speciation will be reviewed.

6.2 PRINCIPLES AND INSTRUMENTS FOR ELEMENTAL SPECIATION USING CAPILLARY ELECTROPHORESIS The basic components of a CE instrument are shown in Fig. 6.1. Typically, a bare fused silica capillary with an inner diameter of 20-100 pm, an outer diameter of 200-400 ]im and a length of 0.2 to 1.0 m is used for CE. The fused silica capillary normally has a polyimide coating on the outside to improve mechanical robustness. In some cases the inside surface of the capillary is coated in order to modify the chemical surface properties. A voltage of 10 to 30 kV is applied between the ends of the capillary. Typically, the inlet side of the capillary is placed in an electrolyte/buffer reservoir following sample injection. The high voltage is applied to the buffer reservoir using a platinum or other relatively inert electrode. On-column detector with capillary flow cell

Regulated HV power supply

r Ni-Chrome electrode

F I I I I L __-

Inlet electrolyteI reservoir j

Electrically isolated housing with safety interlock

\

Outlet electrolyte reservoir

Fused silica capillary 50-100 pm i.d., 50-60 cm long

Fig. 6.1. Basic components of a capillary electrophoresis instrument. 153

If an on-capillary detector is used, the outlet end of the capillary is also placed in an electrolyte/buffer reservoir, connected to ground through an electrode placed into the reservoir solution. If a postcapillary detector, such as ICP-MS or electrospray MS is used, an electrical connection must be made to the outlet end of the capillary using a sheath solution or direct electrical connection. Both laboratory built and commercial CE instruments have been used although not all commercial CE systems are designed to be used with post-capillary detectors. Commercial systems typically provide capillary temperature control, automated sample injection and automated flush cycles after each electropherogram is obtained. 6.2.1 Influence of experimental parameters on migration time The applied voltage, V, produces an electric field gradient (with an average value of VIL for a capillary of length L) that causes positive ions to migrate toward the negatively charged end of the capillary. Negative ions will migrate towards the positively charged end of the capillary. More highly charged ions will tend to migrate more rapidly, as indicated in Fig. 6.2. Many +2 elemental ions have similar equivalent ionic conductances (Table 6.1) and therefore, similar mobilities. Many Injection

Apply 10-30 kV +V

ions migrate under influence of electric field and electroosmotic flow.

Migration and Detection: +V

More highly charged species exit first. Smaller species exit earlier than larger species of equal charge.

Fig. 6.2. Separation of species in an electric field: the basis of capillary electrophoresis separations. 154

TABLE 6.1 Equivalent ionic conductances (104 m2 S mol') for some inorganic cations Ion

Equivalent ionic conductance

Ion

Equivalent ionic conductance

Ion

Equivalent ionic conductance

Li +

38.7

Cu 2+

53.6

Fe 3+

68.0

50.1

2+

Fe

54.0

Cr3 +

67.0

K+

73.5

Mg2 +

53.5

Eu 3 +

67.8

Rb +

77.8

Zn 2+

52.8

Gd 3+

67.3

Na

+

+3 elemental ions have similar mobilities. Singly charged elemental ions have a variety of mobilities. Larger molecular ions have mobilities that depend on their overall charge, size and shape. Two other processes, if present, produce bulk solution flow (electroosmotic flow and laminar flow) that influences the migration time. The migration time, tin, for an ion is given by:

L E( Leff + gt¢of )+ Vam

where L is the length of the capillary, E is the average electric field gradient (V m-'), Veff is the effective electrophoretic mobility of the ion (m 2 V-1 s-), leelf is the electroosmotic mobility and Vlam is the laminar flow velocity (m s-). The origin of electroosmotic flow can be understood by considering the ionic double layer that typically forms near the inner surface of the capillary (Fig. 6.3). Bare fused silica has ionized silanol groups (SiO-) that are protonated to an extent that depends on the electrolyte/buffer pH. Cations from the electrolyte/buffer solution are attracted to the capillary, resulting in a static ionic layer of positive charge density, called the Stern layer or Inner Helmholtz layer. The charge density decreases as the distance from the wall increases. The Stern layer thickness is typically only about 1 nm wide [11]. A more diffuse layer, called the Outer Helmsholtz layer, is formed outside of the Stern layer. This mobile layer has a slight excess of cations. It is a few to 300 nm wide, depending on the extent of silanol group protonation, the voltage applied across the capillary and the electrolyte/buffer concentration and composition. The electrolyte/buffer cations carry waters of hydration. Water molecules in the bulk solution are hydrogen bonded to the waters of hydration around the cations. As the hydrated cations migrate 155

Electroosmotic Flow

~

0)

( 0e (~

e0~

0 e

003 D

ee eE ( Si

si

~ e~ 0 03 (a /

e 0 , o®o 1

0eae'e e

~

Si

S

layer Stern (static)

T0 i

Outer Helmholtz (mobile)

layer Si

Fused Silica Capillary

Fig. 6.3. Diagram of ions in solution near the bare fused silica capillary wall.

towards the more negative potential end of the capillary, the hydrogen bonded water molecules in the bulk solution are dragged along to produce a bulk solution flow. The electroosmotic flow rate depends on the electrolyte/buffer, the pH of the solution and the wall chemistry (which depends on the capillary material and coating, if any). CE separations can be carried out with the direction of electroosmotic flow towards the exit end of the capillary or with the direction of electroosmotic flow towards the inlet end of the capillary. The electroosmotic flow can be in the same direction as the electrophoretic migration of the analyte ions (co-electroosmotic flow mode) or in the opposite direction (counter-electroosmotic flow mode). In contrast to pressure driven separation techniques, CE has a flat flow profile if no laminar flow is induced by the CE interface to a post capillary detector (Fig. 6.4). As a result, CE can provide very high efficiencies, and therefore, often shorter analysis times than ion chromatography or liquid chromatography. The applied voltage gradient (which in turn depends on the applied voltage and the capillary length), the time the analyte spends in the capillary and the inner diameter of the capillary and the induced laminar flow (if any) are the experimental variables that affect migration times and resolution. Several factors can contribute to the analyte CE band width including: the injection volume, Joule heating, 156

T>O

T=O0

T=

R

M

Electoosmotic Flow Profile

n

Dispersion of Injection Plug

T=O

Parabolic Forced Flow Profile

>

T>O

Dispersion of Injection Plug

Fig. 6.4. Flat flow profile characteristic of electroosmotic flow and parabolic flow profile characteristic of laminar flow.

diffusion, concentration overload and laminar flow. These are discussed in more detail below. If the electroosmotic flow velocity is greater than the migration velocity of any analyte, analysis of positively charged, neutral and negatively charged species can be obtained from a single injection. A low or counter-electroosmotic flow can be used to increase migration times and resolution for ions with migration velocities towards the capillary exit that are greater than the electroosmotic flow velocity. However, some ions with slower migration velocities may be lost. Anion analysis would require that the electroosmotic flow be reversed in order to operate in the co-electroosmotic flow mode. Variations in pH and ionic strength, the addition of organic solvents or the addition of surfactants can be used to affect the zeta potential and therefore the electroosmotic flow [11]. Cationic surfactants, such as cetyltrimetylammonium bromide (CTAB) or tetradecyltrimethylammonium bromide (TTAB), can be added to the electrolyte/buffer solution to decrease or reverse the electroosmotic flow rate. However, this will also affect the analyte ion migration velocity. Capillary surface coatings including methylcellulose, carbohydrates, arlypentafluoro compounds, polyacrylamide, polyethylene glycol and C18 hydrophobic groups have been used to affect the electroosmotic flow as well as to reduce analyte-wall interactions. 157

TABLE 6.2 Capillary volumes and injection volumes as a function of capillary inner diameter for a 50 cm long capillary Inner diameter (pm)

Capillary volume (pl)

1% Injection volume (nl)

5% Injection volume (nl)

10

0.04

0.4

2

20

0.16

1.6

8

50

0.98

9.8

49

75

1.5

15

74

100

1.9

20

98

6.2.2 Sample injection Typically the total capillary internal volume is less than a few microliters (Table 6.2). If the sample injection volume is to be less than or equal to 1-5% of the capillary volume, as is often suggested to prevent overloading, sample injection volumes must be on the order of 1 to 100 nl. The sample can be injected into the capillary by hydrostatic, hydrodynamic or electrodynamic modes. 6.2.2.1 Hydrodynamic or hydrostatic injection The sample can be forced into the capillary by inducing a laminar flow by pressurizing the sample vessel or by applying a suction to the outlet end of the capillary (hydrodynamic injection). The injection volume is given by the Poiseuille equation: tPr4 tins 8Lq where P is the pressure drop during injection (N m-2 ), r is the inner radius of the capillary (m), tinj is the injection time (s), L is the length of the capillary (m) and il is the viscosity of the electrolyte/buffer solution. Hydrostatic injection is typically accomplished by placing the inlet end of the capillary into a vessel containing the sample and then raising the sample vessel above the outlet end of the capillary. The injection volume, Vij, can be predicted from: 158

Vi

(pgr2Ahtinj (r

)2

where p is the density of the sample, g is the gravity constant, r is the radius of the CE capillary, Ah is the distance the sample reservoir is raised above the capillary outlet, tj is the injection time, rq is the viscosity of the sample, L is the capillary length. For example, if the reservoir is raised 11 inches above the capillary for 15 s to inject a dilute, aqueous sample, the volume injected into a 50 cm long capillary with a 50 pm i.d. will be approximately 4 nl. For smaller diameter capillaries, higher pressure hydrodynamic injection may be necessary to avoid excessively long injection times. The advantages of hydrostatic and hydrodynamic injection are predictable injection volumes and injection of an amount of sample that is independent of the analyte properties (such as charge or mobility). There is no bias in the sampling of different analytes. 6.2.2.2 Electrokinetic injection In electrostatic injection (often called electromigration injection), the sample moves into the capillary under the influence of an electric field. The inlet end of the capillary is placed into the sample vessel. A voltage is then applied between the sample solution and the outlet end of the capillary for a particular amount of time. The voltage is then turned off and the inlet end of the capillary is taken out of the sample vessel and placed into an electrolyte/buffer reservoir. The quantity of each analyte ion, na, injected into the capillary is theoretically given by:

na

(pa + eof)r

L

2

7

Uinj tin 'c a

where Pa is the analyte ion mobility, ,,eof is the electroosmotic flow mobility, r is the radius of the inside of the capillary, Uinj is the voltage applied during the injection, tin is the injection time and ca is the molar concentration of the analyte. In electrodynamic injection, the amount of analyte injected is dependent on the mobility of the analyte ion. If the analyte has a high mobility, a larger quantity will be injected than if the analyte has a low mobility. If the charge on the analyte is such that it is driven away from the capillary inlet, no analyte will be injected. Neutral species will be carried into the capillary by the electroosmotic flow. Ions with low 159

mobilities have sometimes been observed to exhibit a nonlinear relationship between injection time and the amount injected [12]. The main advantage of electrokinetic sample injection is that it may be possible to preconcentrate the analyte to provide improvements in sensitivity and detection limits of up to a factor of 500. The amount injected can also depend on the conductivity of the sample relative to the CE electrolyte/buffer. The basis, biases and applications of electrokinetic injection for inorganic species have been recently reviewed [13].

6.2.3 Influence of experimental parameters on band dispersion and resolution In order to predict the peak width the following contributing factors must be taken into account [14-16]: longitudinal diffusion, the length of the injected sample plug, Joule heating, concentration overload and if present, laminar flow. Several groups have developed models to predict migration times and band dispersion [17-27]. The simple model by Kinzer et al. [26] added the effect of laminar flow to the models developed previously by Reijenga and Kenndler [17,18]. However, it does not predict peak asymmetries. Mikkers [27] recently published a simple model using a spreadsheet that predicts peak asymmetry. If the sample interacts with the wall of the capillary, additional band dispersion can occur. 6.2.3.1 Longitudinaldiffusion Longitudinal diffusion depends on the migration time (time the analyte species spends in the capillary) and the diffusion coefficient of the analyte species. If all other contributions can be reduced, longitudinal diffusion becomes the ultimate factor limiting resolution. Efficiencies of 50,000 to more than 1,000,000 theoretical plates per meter are then attainable. 6.2.3.2 Injected sample plug The length of the injected sample plug depends on the injection volume and capillary diameter. However, if the conductivity of the sample is less than that of the electrolyte/buffer solution, electrostacking will occur to reduce the effective plug volume containing the analyte after voltage is applied to the capillary [28]. Samples with a lower conductivity than the electrolyte/buffer solution will experience a larger 160

voltage drop/mm across the injected sample volume than across the electrolyte/buffer solution. As a result, the initial migration velocity of the ions within the injected sample plug will be high. As the analyte ions migrate into the background electrolyte the voltage gradient drops abruptly and the analyte migration rate decreases dramatically. As a result, analyte ions will stack at the boundary of the high and low conductivity regions with positive ions on one end of the injected solution plug and negative ions on the other end. The volume containing the analyte after stacking, Vs, will be smaller than the injection volume [29,30]. For small inorganic ions, V, is approximately equal to Vi, the volume of sample injected, times the ratio Is/I, where Is is the ionic strength of the sample and I is the ionic strength of the electrolyte/buffer solution. If the properties of the sample are appropriate, large volume stacking can be used, so that the majority of the capillary is initially filled with sample and then stacking results in preconcentration factors of up to 500 [31-35].

6.2.3.3 Joule heating As current flows through the solution in the electrophoresis capillary Joule heating is produced. Joule heating produces a parabolic flow profile that degrades resolution. In small capillaries (<100 Pm) and at small currents (<40 plA), typical of many CE separations, Joule heating is not a major contributing factor to band broadening. The variance due to Joule heating, Tj is proportional to r6 E4 ' 2A , where r is the inner radius of the capillary, E is the voltage gradient and A is the equivalent conductance of the electrolyte/buffer. The value of 6j is inversely proportional to T4, where T is the absolute temperature. Migration times will also change with temperature because the equivalent ionic conductance depends on temperature. So, maintaining the temperature and using a capillary with a small radius is essential when high conductivity electrolyte/buffer solutions are used. The capillary can be actively cooled to remove more than 5 W of heat [36]. As solutions become heated, gases dissolved in the solution can come out of solution to form a bubble. If heating is sufficient, solvent boiling can occur. The resistance across the bubble is huge compared to the resistance across the electrolyte/buffer solution in the rest of the capillary. As a result, a large fraction of the applied voltage is dropped across the bubble. This can result in a discharge that can cause spurious peaks or even destruction of the capillary ('exploding capillary'!). 161

6.2.3.4 Concentrationoverload The dispersion due to concentration overload (or electromigration dispersion) increases as the effective mobility (or conductivity) of the analyte ion, the analyte concentration and the injection volume increase. In some ways, the concentration overload can be thought of as the opposite of sample stacking. If the voltage gradient across the sample is small compared to the voltage gradient across the electrolyte/ buffer, migration within the sample plug will be slow, degrading resolution. Concentration overload can be avoided by injecting a volume that is a small fraction of the entire capillary and using an electrolyte/ buffer solution with a much higher conductivity than the sample. 6.2.3.5 Laminarflow Electroosmotic flow produces a flat profile (Fig. 6.4) that is in part responsible for the high efficiencies attainable using capillary electrophoresis or electrochromatography (capillary liquid chromatography using electroosmotic flow rather than high pressure induced laminar flow to produce liquid flow). In contrast, laminar flow results in a parabolic flow profile that degrades CE resolution because different portions of the separated sample plug exit the capillary or pass into the detector at different times. The peak width due to laminar flow increases with the radius of the capillary, the square root of the migration time and the laminar flow velocity. Furthermore, the induced laminar flow velocity due to nebulizer suction, discussed below, decreases dramatically as the smaller capillaries are used. 6.2.3.6 Sample-wall interactions Interactions between the sample and capillary wall can result in band broadening as well as sample loss. Attempts are being made to model the sample-wall interactions and their effect on band broadening [37-40]. Sample-wall interactions can be due to chemical adsorption, trapping in the 'natural' double layer discussed above, or trapped in the 'induced' double layer [41,42] that is produced when the voltage is applied between the ends of the capillary. The 'natural' double layer is present as long as the pH of the electrolyte/buffer solution is greater than about 5.3 (the pKa of fused silica) [43]. When a high voltage is applied across the ends of the capillary, a small but significant radial voltage gradient is also produced because of differences in the dielectric strength and conductivity of the electrolyte/buffer solution in the capillary, the fused silica of the capillary walls and the air surrounding 162

the capillary [41,42]. Small ions are particularly susceptible to being trapped in the double layers. The extent of sample ion trapping in the 'induced' double layer can be as high as 90% [42,44]. The trapping can be conveniently assessed by inducing a small laminar flow and then measuring peak areas with and without voltage applied [42,44]. 6.2.4 Selection of experimental parameters There are a large number of experimental parameters that can affect electrophoresis migration times and resolution. Oda and Landers [14] provide a convenient figure showing the effect of 15 experimental parameters on 13 characteristics of importance. 6.2.4.1 Applied voltage As noted above, migration times decrease proportionally to the applied voltage. However, unlike most separation techniques, resolution is not necessary degraded when the analysis time is shortened. In fact, the resolution may improve! The resolution depends on the time spent in the electric field and the electric field gradient. When the voltage is increased, the time spent in the electric field is decreased but the electric field gradient is increased. Furthermore, if longitudinal diffusion is the main source of band broadening, the extent of broadening will be reduced when the migration time is reduced. If the voltage is too high, currents will be high and excessive Joule heating will lead to elevated temperatures, band broadening due to convection flow currents and perhaps formation of bubbles as gases become less soluble in the solution. Typically, a maximum voltage of about 30 kV is used for 0.5 m long capillaries. The typical minimum voltage used is 10 kV. Lower voltages result in long analysis times. Typically, for the analysis of cations, the inlet end of the capillary is held at a positive potential and the exit end is grounded or at a lower positive potential. In this case the electroosmotic flow is towards the exit end. For analysis of anions, often a negative voltage is applied to the inlet and the exit end of the capillary is grounded. In this case, the electric field driven migration of the anions towards the exit of the capillary would be counter to the electroosmotic flow. This could lead to long migration times. The magnitude of the electroosmotic flow can be reduced or the direction of the electroosmotic flow can be reversed by permanent or dynamic coating of the capillary walls, as discussed below. 163

6.2.4.2 Capillary inner diameter Capillaries with a small inner diameter efficiently dissipate Joule heat. Currents are lower due to the smaller cross sectional area, so higher conductivity electrolyte/buffer solutions can be used. This is particularly important for high conductivity samples because the electrolyte/ buffer solution conductivity must be much higher than that for the sample in order to maintain high resolution. Larger capillaries allow larger injection volumes to be used (while maintaining Linj < 0.05 1), often leading to improved concentration based detection limits but poorer resolution. 6.2.4.3 Capillarylength Unlike liquid or gas chromatography, a longer capillary alone may not lead to significantly improved resolution because the voltage gradient will be decreased. Furthermore, analysis times will be increased and longitudinal diffusion will be more extensive so that peak heights will be reduced while peak widths will increase. If the voltage is increased proportionally to the increase in capillary length, resolution will be improved. The practical limit on capillary length appears to be about 100 cm. Typically capillaries at least 20 cm long are used. If the capillary is too short high currents will be generated unless very low conductivity electrolyte/buffer systems are used. The most common capillary length is about 50 cm. 6.2.4.4 Capillarymaterial and coating The inner surface of the capillary directly influences the electroosmotic flow and sample-wall interactions. Bare fused silica capillaries have been most commonly used for elemental speciation. A variety of procedures have been recommended for preparation and regeneration of the electrophoresis capillary in order to strip off any species remaining from prior separations and to obtain a reproducible surface [1]. However, care is required as inappropriate procedures can lead to activation of the surface interactions with sample species [9]. Capillaries are typically conditioned for at least a few hours by filling with the electrolyte/buffer solution prior to use. Some analytes, such as metalloproteins, have a high affinity for fused silica or glass surfaces. A variety of permanent (including neutral polymer, anionic, cationic and hydrophobic) and replaceable coatings have been used to reduce the analyte-wall interactions [15,45]. Surface modification can also be used to affect the electroosmotic flow rate. By reducing the electroosmotic flow rate, analyte ions spend more time in the capillary under 164

the influence of the electric field and resolution is generally improved at the expense of longer analysis times. A variety of capillary materials have also been used for CE although bare fused silica capillaries have been most popular for elemental speciation. 6.2.4.5 Electrolyte / buffer The choice of electrolyte/buffer can be key to successful separations for elemental speciation. The electrolyte/buffer must not change the speciation and must be compatible with the detection technique used. The pH often must be appropriate to maintain the sample speciation. Electrolyte/buffers must not complex with the metal ions if metal-ligand complexation in the original sample is to be monitored. The conductivity or ionic strength of the electrolyte/buffer will determine the current and extent of Joule heating. The extent of sample stacking or peak broadening will depend on the conductivity of the sample relative to the electrolyte/buffer. Ideally, the conductivity of the electrolyte/buffer should be much higher than that of the sample. Increasing the ionic strength of the electrolyte/buffer will increase the thickness of the double layer and in turn reduce the electroosmotic flow rate and possibly decrease analyte-wall interactions. Because of the influence of the electric field on the migration of ions the composition of the electrolyte/buffer may change, or become depleted, over time so it should be replenished often enough to maintain consistent separations. Electrolyte/buffer solution purity must be high enough to prevent high background signals for particular elements of interest. Furthermore, the electrolyte/buffer solution should be filtered and degassed before use to prevent capillary plugging and bubble formation (that could lead to arcing, spurious peaks and capillary destruction, as discussed above). Peak asymmetry will vary depending on the mobility of the analyte ions relative to the co-migrating electrolyte/buffer ions. Optimum peak symmetry will be obtained when the mobilities of the analyte and comigrating electrolyte/buffer ions are similar. Otherwise, extensive peak fronting or tailing may be observed depending on whether the mobility of the analyte ion is much higher or much lower than the comigrating electrolyte/buffer ion. 6.2.4.6 Injection volume The appropriate sample injection volume depends on several factors including the capillary inner diameter and the conductivity of the sample solution relative to that of the electrolyte/buffer solution. The 165

optimum injection volume may also depend on trade offs between concentration based peak area detection limits and the resolution required. 6.2.4.7 Optimizing injection volume, capillarydiameter and electrolyte / buffer conductivity As noted above, optimum resolution is obtained when the sample conductivity is far less than that of the electrolyte/buffer. However, the maximum conductivity of the electrolyte/buffer solution that can be tolerated depends on the generation and dissipation of Joule heat. The higher the conductivity of the sample the smaller the injection volume that can be used while maintaining the capillary diameter and electrolyte/buffer conductivity. If a smaller i.d. capillary is used, then a higher conductivity electrolyte/buffer can be used to reduce concentration overload band dispersion. Figure 6.5a shows an electropherogram obtained from a 50 nl injection of a multi-element sample with 6 species each at a concentration of 1 pg/ml into a 100 pm i.d. capillary [46]. The electrolyte was 2.7 mM CaCl2 . The injection volume could be increased to 500 nl with only a slight increase in peak widths (Fig. 6.5b). However, when 50 nl of a 100 pg/ml sample is injected, peak distortion and broadening is observed (Fig. 6.5c). The situation becomes much worse when a 500 nl injection volume is used (Fig. 6.5d). However, when 5 nl of the 100 pg/ml solution is injected into a 30 pm i.d. capillary with 26 mM CaCl2 electrolyte, good resolution is obtained again (Fig. 6.5e). With a similar injection volume and electrolyte concentration, the four species shown are still resolved when a 1000 pg/ml analyte solution is injected (Fig. 6.5f). Note that the loss of resolution due to concentration overload is worst for species with mobilities that are either much higher (such as K+) or much lower (such as Li +) than the electrolyte co-migrating ion (Ca2+). When a smaller injection volume is used signal sensitivity is lower.

6.3 DETECTORS FOR ELEMENTAL SPECIATION BY CE 6.3.1 On-capillary detection The advantage of on-capillary detectors is that the detector will not affect the separation process, such as by inducing a laminar flow in the 166

a 40 -

1 ppm Cs+ Y3

50 nL

Co 2

2 30-

I

:i

, 20-

69dialft"

Li

10-

0

1;1 I

I

Ir

0

Migration time (s)

400

0

Migration time (s)

400

Fig. 6.5. Effect of sample conductivity, injection volume, capillary diameter and electrolyte/buffer concentration on separation of inorganic ions. (a) 50 nl injection of 1 g/ ml analyte solution. (b) 500 nl injection of 1 g/ml analyte solution. (c) 50 nl injection of 100 pg/ml analyte solution. (d) 500 nl injection of 100 g/ml analyte solution. (e) 5 nl injection of 100 pg/ml solution. (f) 5 nl injection of 1000 pg/ml solution. A 100 pm i.d. fused silica capillary was used with a 2.7 mM CaCI2 electrolyte for electropherograms shown in (a), (b), (c) and (d). A 30 pm i.d. fused silica capillary was used with a 26 mM CaC12 electrolyte for electropherograms shown in (e) and (f). The Li signal is shown on a different scale (not shown) on each plot. The Co signal was multiplied by 3 before plotting. Data source: [46]. 167

capillary. Also, the electrical connection to the outlet end of the capillary can be easily and conveniently made through a reservoir into which the end of the capillary is placed. 6.3.1.1 Indirect LV detection The most widely used detector for capillary electrophoresis of inorganic ions and elemental speciation is indirect UV absorption [47-49]. Most metal ions and metal containing species do not absorb UV light. However, indirect UV absorbance detection can be used by adding a UV absorbing species to the CE electrolyte/buffer. Alternatively, metal ions could be complexed with an organic ligand to form a UV absorbing species either before injection or post-capillary. Indirect UV absorbance detection depends on measuring decreases in the local concentration of the chromophore species that results due to displacement. The displacement mechanism can be due to simple conservation of volume or charge displacement. Low detection limits using indirect UV absorbance detection, or other indirect detection schemes, require a large transfer ratio (amount of chromophore species displaced per concentration of analyte) and a very stable background signal. Optimization and prediction of transfer ratios for both cations and anions has been discussed [50-541. As the detection limit is approached, extremely small differences in absorbance must be detected. 6.3.1.2 Direct on-capillarydetection by electrochemical detection Electrochemical detection provides advantages including high sensitivity, low cost and selectivity. Conductometric, amperometric and potentiometric detectors for capillary electrophoresis have been recently reviewed [55,56]. Normal conductivity detection provides detection limits on the order of 10 -5 M [55]. Background signals can be depressed by removing electrolyte/buffer ions using weak acids or bases. These become non-ionic when they come in contact with an ion-exchange membrane. This suppressed conductivity detection can supply 10- 7 M detection limits [55]. Suppressed conductivity systems are effective but only for a limited number of electrolyte/buffer systems [56]. Conductivity detection may have the potential to be a more sensitive universal detector than indirect UV absorbance [55,56]. Its main limitation is susceptibility to fouling. Ion-selective microelectrodes can be used for potentiometric detection, providing some selectivity. Speciation of transition metals has been accomplished with amperometric detection [56]. The use of 168

electrochemical detection for elemental speciation using capillary electrophoresis is still fairly rare. In some cases this has been due to inadequate detection limits or a lack of ruggedness and changes in sensitivity over time. 6.3.1.3 Direct on-capillary detection by laser induced fluorescence For a limited number of specific analytes, laser induced fluorescence (LIF) detection has been used for CE elemental speciation detection. The interactions between metals and humic substances were investigated by CE with LIF detection [57]. This requires species that fluoresce or derivatization to form species that fluoresce. 6.3.1.4 UV absorptiondetection following complexation Complexation reactions have been used either before sample injection or in post-capillary derivatization to produce species that can then be detected by UV absorption or laser induced fluorescence. For example, capillary electrophoresis was used to measure Fe(II) and Fe(III) following selective complexation with a mixture of o-phenanthroline and EDTA [58]. The concentrations of metal-ligand complexes were then measured following capillary electrophoresis separation using direct UV detection. When only EDTA was added, total Fe could be measured by direct UV absorption. In this case, the complexation provides a means to detect the species of interest as well as preventing changes in the relative concentrations of Fe(II) and Fe(III) during the separation.

6.3.2 Post-capillary detectors Elemental and molecular mass spectrometry detectors can provide specificity, species identification and sensitivity that is typically not attainable using on-capillary detectors, such as direct or indirect UV absorption. Inductively coupled plasma mass spectrometry [8], plasma optical emission spectroscopy and electrospray or ion spray mass spectrometry have been used for elemental speciation with capillary electrophoresis. The sample must be physically transported from the CE capillary to the detector for mass spectrometric detection or for atomic spectroscopy using inductively coupled plasma or microwave plasma sources. As a result, the interface between the CE and the detector is critical. 169

6.3.2.1 Interface considerations Four factors must be considered to interface CE with post-capillary detectors. There must be a means to make an electrical contact with the electrolyte/buffer at the outlet end of the capillary. This is most commonly done using a conductive sheath flow solution. The sweep-out time for any dead volume in the interface must be small compared to the CE peak width or resolution will be lost. The sample must be transported to the detector as efficiently as possible in a form that can be efficiently converted into analyte signals by the detector. The post-capillary detectors listed above generate signals that depend on the mass of analyte delivered to the detector, not the concentration of the solution. The CE injection volume is very small, so concentration based detection limits typically will be somewhat poorer than when these detectors are used with larger sample volumes. In order to maximize sensitivity a fine aerosol must be delivered into the detection instrument with high efficiency. The effect of the interface on the electrophoretic separation in the capillary must be carefully controlled. If the nebulizer or sprayer used to form an aerosol from the solution exiting the capillary produces a suction, laminar flow can be induced in the capillary. Because the electroosmotic flow is so low and because laminar flow produces a parabolic rather than flat flow profile, even small amounts (<1 p1/min) of induced laminar flow can degrade CE resolution. Conversely, laminar flow can be advantageous in some cases [26]. Positive ions, negative ions and neutral species can be measured from a single injection if laminar flow is induced. Analysis times can be shortened when high efficiency is not needed. In some cases, laminar flow away from the detector can lead to improved resolution [26]. 6.3.2.2 Inductively coupled plasma optical emission and mass spectrometry The combination of ICP-OES and ICP-MS for rapid elemental speciation was described in 1995 by Olesik et al. [44]. Several groups have developed interfaces and shown CE-ICP-MS in particular to be a promising technique for elemental speciation. 6.3.2.2.1 Interfaces The most widely used interface for CE-ICP-MS is based on a sheath flow system and a concentric nebulizer, similar to that shown in Fig. 6.6, and first described by Lu, Bird and Barnes [59]. Several systems 170

Fused silica capillary Stainless steel union

Teflon union Stainless steel tubing

Argon inlet

Sheath flow solution delivered by syringe pump

Fig. 6.6. Sheath flow type interface for CE-ICP-MS.

based on similar concepts have been reported with a variety of pneumatic nebulizers including the Meinhard® HEN [26], modified Meinhard® [60], direct injection [61], Cetac MCN [62,63], cross-flow [63,64] and laboratory built concentric nebulizers [64,65]. It is interesting that there has been such a focus on different nebulizers when the analyte transport rate is much less dependent on the nebulizer type than on the rate of total solution delivered to the nebulizer [66], as discussed below. Sheath flow systems were previously used for interfacing CE and electrospray MS [67]. The outlet end of the capillary is inserted into the center tube of a concentric nebulizer, as shown in Fig. 6.6. For conventional concentric nebulizers, like the Meinhard® TR-30, a capillary with an outer diameter around 300 ipm can be inserted well into the center tube of the nebulizer. For microconcentric nebulizers (which have a small internal volume), the center tube of the nebulizer is narrower so the electrophoresis capillary can only be inserted into the nebulizer to the point where the center tube begins (as shown in Fig. 6.6). The sheath solution is introduced into a tee connector through which the electrophoresis capillary passes. The sheath solution then flows concentrically around the electrophoresis capillary until it mixes with the solution exiting the outlet end of the capillary. The sheath flow solution serves up to three functions. First, an electrical connection, typically ground, is made through the sheath flow solution to the outlet end of the electrophoresis capillary. Second, the use of a liquid sheath flow reduces the sweep out time through the nebulizer dead volume. Third, by controlling the sheath flow rate, laminar flow due to nebulizer suction can be eliminated, controlled in a direction towards the electrophoresis capillary outlet or induced in a direction away from the electrophoresis capillary outlet. 171

The main disadvantage when a sheath liquid is used is that the transport efficiency of aerosol from the nebulizer, through the spray chamber and into the plasma decreases as the total volume of solution delivered to the nebulizer is increased [66,68,69]. For example, using a concentric, pneumatic nebulizer and a Scott-type double pass spray chamber, the analyte transport efficiency decreased from 50% at 10 pl/min, to 13% at 100 pl/min to 2.5% at 1000 pi/min [66, 69]. There is only a slight dependence of analyte transport efficiency on concentric nebulizer type (Meinhard® TR-30, Meinhard® HEN or Cetac MCN) when comparisons are made at a particular nebulizer gas flow rate and sample uptake rate for all nebulizers. The decrease in analyte transport efficiency with increasing solution flow rate is likely due to the increasing frequency of droplet-droplet collisions and coagulation in the spray chamber. The large coagulated droplets are then lost. Therefore, from a sensitivity standpoint, low sheath flow rates are desirable. However, sheath flow rates of 50 pl/min or more are often required to eliminate induced laminar flow, as discussed below. The direct injection nebulizer (DIN) provides 100% aerosol transport efficiency into the ICP because the aerosol is directly into the plasma without passing through a spray chamber [70-72]. Liu et al. [61] described the use of a DIN based interface for CE-ICP-MS. A 15 Pl/ min make up or sheath flow was used because the minimum DIN flow rate is about 10 pl/min. Detection limits for a variety of elemental species were better than 1 ng/ml with Tl + having a detection limit of 7 pg/ml. The concentration based detection limits were typically about a factor of 15 higher than for continuous sample introduction. It is likely that the recently introduced direct injection high efficiency nebulizer (DIHEN) [73] will also be used for CE-ICP-MS. Because the DIHEN is typically operated at gas flow rates as low as 0.2 /min, the induced laminar flow rate may be lower than for concentric, pneumatic nebulizers operated at much higher gas flow rates. Lu and Barnes [74] reported on the use of an ultrasonic nebulizer interface for CE-ICP-MS using a sheath flow to make an electrical connection to the outlet end of the capillary. Because an ultrasonic nebulizer does not produce a suction in the sample inlet tube, no laminar flow is induced in the electrophoresis capillary. The sensitivity was reported to be 3-8 times higher than when a concentric pneumatic nebulizer was used. However, the noise also increased so that improvements in detection limits were less than a factor of three. 172

A sheathless interface was described in which the electrophoresis capillary was inserted completely to the end of the center tube of a concentric nebulizer [44]. Aerosol was generated directly from the end of the capillary, eliminating any dead volume before nebulization. An electrical connection was made using a silver paint on the outside of the capillary. Analyte transport efficiencies from the nebulizer into the ICP are greater than 60% [42]. The disadvantage of this interface is that a large (1-pl/min) laminar flow was induced in the electrophoresis capillary. While this might be overcome by applying a negative pressure, electrical connection to the solution at the exit end of the capillary might be more difficult at sub-pl flow rates because the capillary tip might not be well wetted. Mei et al. [75] described an interface that used a Y-shaped quartz connector with tapered inlets to connect the electrophoresis capillary output and the output from an auxiliary electrolyte/buffer reservoir to a nebulizer. Some control of the induced laminar flow rate was achieved selecting the inner diameter and length of the auxiliary capillary relative to the electrophoresis capillary. For elements that form volatile hydrides, the analyte can be introduced as a gas. Two different groups have described hydride generation based interfaces for CE-ICP-MS and CE-ICP-OES [76-78]. In addition to high analyte transport efficiency into the plasma, the hydride generation systems have the advantages of not introducing water or species that do not form volatile hydrides into the ICP. As a result, concomitant species matrix effects and signals from polyatomic ions such as ArO + and ArH + , ArCl1 are greatly reduced. If laminar flow is eliminated, high efficiency separations can be obtained by CE-ICP-MS. Efficiencies as high as 400,000 plates/m have been obtained, even for inorganic ions with high mobilities. According to CE models, it should be possible to obtain peaks significantly less than 1 s wide if the conductivity of the electrolyte/buffer solution is very high compared to the sample. Our experience has been that peak widths of about 1 s are the narrowest we have observed. It is not yet clear if this is due to a limitation in the CE or perhaps the result of broadening due to the spray chamber. 6.3.2.2.2 Assessment and control of laminar flow Most pneumatic nebulizers produce a suction that draws solution into the nebulizer and towards the nebulizer tip. This can induce a laminar flow in the electrophoresis capillary that produces a parabolic rather 173

than flat flow profile. As a result, electrophoretic resolution is typically degraded if laminar flow is present. However, laminar flow towards the capillary outlet can allow measurement of negative and positive ions from one sample injection. The magnitude of the nebulizer induced suction depends on several nebulizer design parameters including the area of the ring through which the nebulizer gas flows, the distance between the liquid carrying center tube and the gas ring, the shape of the center tube and the protrusion or indentation distance between the end of the nebulizer center tube and the gas ring. The magnitude of the suction increases as the nebulizer gas flow rate is increased. When the sheath flow rate is low, suction in the center tube of the nebulizer due to the Venturi effect as the nebulizer gas exits the nebulizer produces a laminar flow in the electrophoresis capillary towards the detector. If the sheath flow rate is high, a backpressure is produced in the center tube of the nebulizer and laminar flow is induced in the electrophoresis capillary towards its inlet end. If the sheath flow rate is just high enough to exactly balance the suction produced by the exiting nebulizer gas, no laminar flow is induced in the electrophoresis capillary. The induced laminar flow rate depends on the position of the electrophoresis capillary within the center tube of the nebulizer [59]. The induced laminar flow rate also decreases dramatically as the inner diameter of the electrophoresis capillary is reduced relative to the area around the outside of the electrophoresis capillary through which the sheath solution flows. Figure 6.7 shows the effect of the sheath flow rate on the induced laminar flow rate for two capillaries with different inner diameters but similar outer diameters. The sheath flow rate necessary to eliminate laminar flow is independent of the electrophoresis capillary inner diameter. However, the induced laminar flow is almost two orders of magnitude lower for the 25 m i.d. capillary than the 100 m capillary for similar sheath flow rates. The magnitude of induced laminar flow is easily measured by injecting a sample and measuring the migration time with no voltage applied across the capillary. The induced laminar flow rate can be calculated from: Tr2L U lamn

174

tlll_

where r is the inner radius of the electrophoresis capillary, L is the length of the capillary and to,, is the migration time with no voltage applied. Figure 6.7 shows the measured induced laminar flow rate as a function of sheath flow rate for a typical Meinhard® HEN microconcentric nebulizer operated at a nebulizer gas flow rate of 0.8 1/min. Because the relationship between the induced laminar flow rate and the sheath flow rate is linear, the behavior can be extrapolated to zero induced laminar flow rate in order to determine the sheath flow rate that will eliminate laminar flow in the electrophoresis capillary. Considering how straightforward it is to measure the induced laminar flow rate and to estimate the sheath flow rate required to eliminate induced laminar flow, it is surprising that many researchers do not seem to experimentally determine these. For some nebulizers, high (>100 pl/min) sheath flow rates are needed to eliminate induced laminar flow. An alternative means to control laminar flow is to apply a negative pressure to the inlet reservoir container [59]. Conveniently, some commercial CE systems include a means to apply a negative pressure (originally intended to be used to flush the capillary between sample injections). By applying 0.05s .0

4

30 m ID

0.04 d 0.03

I

0.02

3 -

B 0.01

'L

100 pm ID

2 O

153 pLlin

0

25 50 75 100 125 150 Sheath Flow Rate (pL/min)

159

CI

pIL/min

E

30 pm ID 0 0

40

80

120

160

200

Sheath Flow Rate (pL/min) Fig. 6.7. Induced laminar flow rate versus sheath flow rate for two different i.d. capillaries. The inset shows an expanded scale view of the laminar flow rate as a function of sheath flow rate for a 30 pm i.d., 0.5 m long capillary. The experimentally measured balance points (sheath flow rate that eliminates induced laminar flow) were 153 and 159 pl/min for the 30 and 100 pm i.d. capillaries, respectively. Data source: [46].

175

negative pressure to the inlet reservoir, lower sheath flow rates can be used, thereby improving the transport efficiency from the nebulizer to the plasma, as discussed above. 6.3.2.2.3 Sensitivity and detection limits Sensitivity (signal/concentration) and detection limits in ICP-OES and ICP-MS depend on the mass (or number of moles) of analyte delivered to the plasma per unit time. The small (nl) sample volumes typically used in CE can limit ICP-OES and ICP-MS concentration based sensitivity and detection limits. This can be one of the main limitations of CE-ICP-OES and CE-ICP-MS as an elemental speciation technique. ICP-MS has been more widely used for CE detection than ICP-OES because ICP-MS detection limits are generally up to 1000 times lower than ICP-OES detection limits for continuous, 1 ml/min sample introduction. Concentration based sensitivity and detection limits depend on the sample injection volume, the electrophoretic peak width, the transport efficiency of analyte from the nebulizer through the spray chamber and into the ICP and any post-capillary band broadening due to finite dead volume sweep out times in the solution interface or spray chamber. Two common misconceptions seem to have confused several researchers who have developed CE-ICP-MS interfaces. Some claim that transport efficiency for pneumatic nebulizers/spray chambers is poor so ultrasonic, direct injection or microconcentric nebulizers are needed at low sample uptake rates. However, this statement is most likely based on comparing the analyte transport efficiency of one of the 'high efficiency' nebulizers at a low solution uptake rate (<100 Pl/min) to that observed for a 'standard' nebulizer operated at a high solution uptake rate (1000 Vl/min). In fact, Meinhard® TR-30, HEN and Cetac MCN microconcentric nebulizers all provide similar transport efficiencies when operated at similar solution flow rates and nebulizer gas flow rates [66]. Furthermore, analyte transport efficiencies of 20-80% can be obtained using a pneumatic concentric nebulizer if the solution flow rate is low, as discussed above. A second misconception is that the sheath flow 'dilutes' the sample resulting in degraded detection limits. The sensitivity depends on the mass of analyte transported into the plasma, not the concentration of the solution introduced. If the analyte transport efficiency from the nebulizer through the spray chamber to the plasma and the signal per mass of analyte entering the plasma were constant, the sensitivity 176

would be independent of the sheath flow rate. However, as noted above, the analyte transport rate and the ICP signal decreases quickly as the total solution flow rate to the nebulizer is increased [66,68,69,79,80]. Therefore, the use of a sheath flow does not cause a decrease in sensitivity due to dilution as some have suggested but rather due to a decrease in analyte transport efficiency through the spray chamber. If the analyte transport efficiency was 20% (typical of a pneumatic nebulizer and double pass spray chamber with a total solution flow rate to the nebulizer of 50 pl/min), then only a factor of 5 improvement in detection limits would be possible by using a different spray chamber and nebulizer system that produced 100% transport efficiency. However, if higher sheath flow rates are used, the analyte transport efficiency will be lower. In this case, a sample introduction system such as the High Efficiency Sample Introduction System [81] that appears to provide nearly 100% analyte transport efficiency at uptake rates up to about 300 il/min could lead to improvements in analyte transport rate of up to a factor of 10. Detection limits provided by this system would theoretically be independent of the sheath flow rate. The detection limits that should be attainable for CE-ICP-MS can be estimated beginning from detection limits obtained using continuous sample introduction for total elemental analysis. Assume the detection limit for conventional, continuous sample introduction and ICP-MS detection is 1 part per trillion (pg/ml). Assume a typical analyte transport efficiency is about 2% at a sample uptake rate of 1 ml/ min. Then 0.02 pg (20 fg) of analyte are delivered into the ICP each minute or 0.333 fg/s. Consider a CE sample injection volume of 50 nl, a peak width of approximately 10 s and an analyte transport rate of 33% (rather than 2% because of the lower total solution flow rate to the nebulizer). At the detection limit 3.333 fg of the analyte must enter the plasma in 10 s. Assuming a transport efficiency of 33%, 10 fg of sample must be delivered to the nebulizer in 10 s. Therefore, the concentration based detection limit would be 10 fg/50 nl or 10 ng/50 ml, equivalent to 0.20 ng/ml or 200 parts per trillion. This predicts concentration based detection limits that are a factor of 200 poorer than continuous sample introduction at 1 ml/min, assuming no loss of sample in the electrophoresis capillary. If the injection volume could be increased without increasing the peak width (such as by sample stacking) or the peak could be made narrower for the same injection volume, the detection limits would improve proportionally. 177

6.3.2.2.4 Additional detection issues in ICP-MS Sector based ICP-MS instruments have two potential advantages for CE detection: high sensitivity when used in a low resolution mode or high resolution (but with typically a factor of 20 lower sensitivity when the resolution is increased from about 250 to 10,000). Typical quadrupole ICP-MS instruments provide sensitivities of 10-50 million counts/ s/ppm solution when the sample uptake rate is 1 ml/min and background count rates of a few counts/s when no contamination is present and there are no spectral overlaps. Sector based ICP-MS instruments, when used in a low resolution mode, can provide sensitivities that are 10-50 times higher with background count rates that are up to a factor of ten lower [82]. Newly introduced quadrupole ICP-MS instruments that have reaction/collision cells also reportedly produce lower limiting background count rates, well below 1 c/s, and therefore, improved detection limits. For many elements of interest, particularly those with masses below 81 such as P, Fe, Cr, As, Sn and Se, molecular ion spectral overlaps can severely degrade ICP-MS detection limits. High resolution mass spectrometers can overcome some of these spectral overlaps although the sensitivity will decrease significantly compared to the low resolution mode. Newly introduced mass spectrometers from PE-Sciex and Micromass that use a reaction cell to remove particular elemental or molecular ion spectral overlaps [83,84] promise to be particularly useful to improve detection limits for many of these elements. Detection limits also depend on the fraction of time each analyte mass is measured. If many elements are to be monitored, the detection limits will be degraded somewhat compared to single ion monitoring. If the peaks are very narrow (1 s, for example) there could be problems in monitoring many elements. In this case, time of flight mass spectrometry, which monitors all mass simultaneously has potential advantages [85]. However, currently the duty cycle for time of flight mass spectrometry is also less than 100% and signal/noise ratios tend to be somewhat poorer than for commercial quadrupole based mass spectrometers. The main limitation of the element selective detectors is that they do not provide direct identification of the detected species. A combination of migration times and elemental composition is used. It may be necessary to spike the sample with known species in order to identify the molecular composition of the species.

178

6.3.2.3 Electrospray and ion spray mass spectrometry Electrospray (ES) and ion spray (IS) mass spectrometry can provide identification of molecular ions by transferring ions from solution into the gas phase and then measuring their mass spectra. ES-MS and ISMS can be used for elemental speciation without a prior separation [86,87]. However, the spectra can be quite complex due to solvent clustering and gas phase reactions so that analysis of complex mixtures can be difficult. Therefore, ES-MS or IS-MS detection combined with a separation technique, such as CE, is potentially very attractive. In fact, ES-MS and IS-MS are widely used with capillary electrophoresis for analysis of organic compounds [88]. The main limitations of ES-MS and IS-MS as a detector for CE are poorer concentration based detection limits than ICP-MS, strongly compound dependent sensitivities and severe reductions in sensitivity when some electrolyte/buffer solutions are used. Three different types of interfaces [88,89] have been used: coaxial sheath-flow [67,90], liquid junction [91] and sheathless [92,94]. The coaxial sheath-flow interface (Fig. 6.8a) is similar in concept to that used to couple CE and ICP, discussed above, and is the most widely used interface. In the liquid junction interface (Fig. 6.8b) electrical contact to the exit end of the electrophoresis capillary is made through a solution reservoir that surrounds the electrophoresis capillary and a transfer capillary used to carry the analyte to the electrospray or ion spray needle tip. The two capillaries must be precisely aligned and with a gap of 10-20 pm [88]. If the capillaries are too close, sufficient make-up liquid will not be drawn into the transfer capillary by the suction produced by the nebulizer and a laminar flow will be induced in the electrophoresis capillary. If the capillaries are too far apart analyte ions will be lost into the make-up reservoir due to diffusion. Most sheathless interfaces (Fig. 6.8c) use a sharply tapered, metal coated capillary tip [93,95] in order to make an electrical connection to the electrophoresis capillary and to reduce the potential required to spray solutions with conductivities and surface tensions characteristic of aqueous solutions. Other sheathless interfaces that use an in-capillary electrode have also been reported [94]. The potential advantage of the sheathless interface is higher sensitivity. The disadvantages include unmatched flow rates for optimum CE and electrospray, particularly if the electroosmotic flow rate is low and the potential

179

(a)

I

Separation Capillary

- I ___ _

ESI Powe_ Supply

ItI I

Sheath Sheath Liquid Gas

(b)

Separation Capillary--

I {. Liquid Junction

Sheath Gas (c)

Separation Capillary I ESI Power Supply

Gold-Tipped I Capillary

It

Sheath Gas

Fig. 6.8. Interfaces for capillary electrophoresis-electrospray ionization mass spectrometry. (a) Concentric sheath flow interface. (b) Liquid-junction based interface. (c) Sheathless interface. From: [89]. Reprinted by permission of John Wiley & Sons, Inc.

problems due to the electrophoresis electrolyte/buffer without mixing with more volatile sheath-flow solutions. 6.3.2.4 Proton induced X-ray emission (PIXE) Element selective CE detection can be obtained by PIXE [96-98]. A high energy (1700 keV) proton beam is generated using a Van de Graaff accelerator. The beam is collimated and directed through an 'ion window' in a fused silica electrophoresis capillary formed by etching 180

the capillary wall to a thickness of less than 10 pm. An X-ray detector is placed within 5 mm of the capillary. Detection limits for species containing a variety of elements including As, Cu, Fe, Ni, Pb, Rb, Y and Zn were in the 10 -5 M range [97].

6.4 APPLICATIONS OF CAPILLARY ELECTROPHORESIS FOR ELEMENTAL SPECIATION Capillary electrophoresis is still in the early stages of development compared to the use of gas chromatography, liquid chromatography and ion chromatography for elemental speciation. However, in the 1990s there has been a rapid growth in the use of CE for speciation. Below are selected applications of CE for elemental speciation intended to illustrate the analysis capabilities and some of the limitations. 6.4.1 Charge (oxidation) state speciation Because separation in CE is based on the charge on the analyte, it would seem to be ideally suited for rapid speciation of species such as CrO42- and Cr 3 +, Fe2 + and Fe3+, Sn 2+ and Sn4 + , Hg2 + and Hg ° , VO2+ and V 2 +. Cr(III) is an essential nutrient element for glucose metabolism while Cr(VI) is carcinogenic [99]. One of the potential problems in using CE for chromium speciation is that these small ions have high mobilities in opposite directions in an electric field. As a result, the electroosmotic flow rate is too small to carry the ion that moves away from the exit end of the electrophoresis capillary under the influence of the electric field to the exit end. One approach to solve this problem so that the two chromium species can be measured from a single injection is to form a stable, kinetically inert, negatively charged complex of Cr(III) with 1,2-cyclohexane-diaminetraacetic acid (CDTA) [100], EDTA or DTPA [101]. Direct UV absorption detection can then be used to obtain detection limits in the 10 -6 M concentration range [100, 101] in electroplating solutions and pharmaceutical preparations with analysis times of about 10 min. Mo(VI) can be used to react with Cr(III) to form a heteropolyanion and then it can be separated from Cr(VI) anion species using CE [102]. Another approach to analyze positively and negatively charged chromium species from a single injection is to use a positive laminar 181

flow at a large enough rate to overcome the migration of the ion being driven away from the exit end of the electrophoresis capillary [26,44]. This will result in a loss of electrophoretic resolution that could lead to peak overlaps that would be problematical if a nonselective detector was used. However, if an element selective detector is used this would not be a problem because the negative and positive chromium ions are easily separated. Analysis times can also be reduced to less than 2 min. Fe(II) and Fe(III) have been measured using CE following complexation with o-phenantroline and EDTA [58,101] and direct UV detection. Detection limits were less than 1 ppm for an injection volume of 22.6 nl. It was found that the addition of an excess of EDTA alone to the sample caused rapid oxidation of Fe(II) to Fe(III). The complexation constant for Fe(II) with o-phenantroline is 1021-2 while for EDTA it is 10143. Fe(III) has complexation constants of 10144 and 10316 for ophenantroline and EDTA, respectively. So, o-phenantroline complexes selectively with Fe(II) while EDTA complexes selectively with Fe(III) when a mixture of o-phenantroline and EDTA is used. This prevents changes in the relative amounts of Fe(II) and Fe(III). Peak area signals had a precision better than 5% [58]. CDTA can be used in place of EDTA so that analysis times of less than 2 min can be attained with detection limits for Fe(II) and Fe(III) each below 0.1 ppm [103]. This approach was used to analyze tap water and ground water samples as well as atmospheric aerosols [103]. Fe(II) and Fe(III) may be separated directly by CE [44]. However, further investigation with a variety of real samples is necessary in order to assess potential changes in Fe(II) and Fe(III) speciation during the analysis. V(IV) and V(V) species have also been measured without complexation [104] and following complexation with EDTA, CDTA or DTPA [101,105]. Detection limits were in the range of 0.1 to 0.4 pg/ml. 6.4.2 Arsenic and selenium speciation The toxicity of arsenic varies widely, dependent on the species present. Speciation of arsenic in humans depends both on the species taken in and metabolism in the body [106]. Inorganic arsenic, arsenate (AsO4 3-) and arsenite (AsO2-), are highly toxic. Arsenate is reduced to arsenite in the body and then to monomethylarsonate (MMA) and dimethylarsonate by two different enzymes, which reduces their toxicity [106]. MMA and DMA are about 1000 times less toxic than the inorganic 182

arsenic species. Arsenobetaine and arsenochlorine are commonly found in seafood and are relatively non-toxic. A variety of arsenic compounds are used as anti-fungal agents, pharmaceuticals, herbicides and in semi-conductor processing. Selenium intake is essential but excess intake can cause toxic reactions. Low concentration of selenium in blood has been identified as a risk factor for several diseases including heart disease, cancer, rheumatoid arthritis, muscular dystrophy and cystic fibrosis [107]. However, the distribution of selenium in selenoproteins varies among different species and in tissues within the same species [107]. Furthermore, there appears to be a 'hierarchy' of importance of selenoproteins. A marginal deficiency in selenium intake will then affect the levels of some selenoproteins but not others [107]. Therefore, the levels of a number of selenoproteins must be monitored. A variety of selenium containing species are present in the environment, natural foods and supplements. Selenomethionine is present principally in plant foods while selenocystine is present in food from animals [108]. Selenite and selenate are often used as dietary supplements. Rapid, reliable methods for arsenic and selenium speciation are needed. As a result, there has been a great deal of activity in developing CE methods using indirect UV absorption, direct UV absorption, element specific and molecular mass spectrometry detectors for arsenic and selenium speciation. Nearly 100 publications focused on the development or application of CE for arsenic or selenium speciation. 6.4.2.1 Arsenic and selenium speciation using nonspecific detection Wildman et al. [109] reported the measurement of inorganic arsenic in urine using capillary electrophoresis in 1991. Arsenite (AsO,-), arsenate (As0 4 3-), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) were separated by CE using an acidic phosphate buffer (pH 4.5-6.5) and detected using direct UV absorption at 190 nm [110]. The authors noted the advantages of CE: relatively simple equipment, a short analysis time (15 min), high efficiency and low mass detection limit (40 pg for arsenate). Arsenite, arsenate, monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and phenylarsonic acid were separated using CE and detected at the mg/l level in spiked drinking water [111]. Mean recoveries were between 97 and 104%. Caruso and co-workers [112] reported the detection of arsenite, arsenate, DMA and MMA from coal fly ash extracts using CE with indirect UV detection and an analysis time of less than 6 min (Fig. 6.9a). In this case a 183

(a) 0

o

1 -

34 -0.005

2

5 I

1

4 5 Time (min)

6

7

(b) Ouu

L-""`

3

16000 -

2 APAO 3 PAO 4 DMA 5 DPAA 6 APAA 7 PAA 8 MA 9 As(V) 10 PAS

14000-

12000 6

2

10000-

4

8000-

C

6000-

5 7 10

1

9

U. 4

4000 -

2000

I

5

6

i

.

I

7

8

9

10

11

I

12

I

13

I

14

15

16

. I

17

Migration Time [mini

Fig. 6.9. Arsenic speciation. (a) Electropherogram of four arsenic species at 10 pg/ml As each using indirect UV absorption detection at 274 nm (112). The peaks are: 1: unidentified, 2: As(V), 3: MMA, 4: DMA, 5: As(III). (b) Electropherogram of seven arsenic species at 15 pg/ml As each using direct UV absorption detection [116].

chromate buffer was used with NICE-Pak OFM Anion-BT to modify the electroosmotic flow. Chromate was used as the indirect UV absorber. Absolute detection limits were 5-16 pg. The injection volume was 8 nl, so concentration based detection limits were 0.6-2 g/ml. Schwedt and Rieckhoff [113] compared ion chromatography and CE for the separation of thio- and osothio-arsenates. Decomposition of 184

these species during separation depended on pH, temperature and whether a coated or uncoated capillary was used. They were able to achieve higher efficiencies, better separations and better reproducibility using CE compared to ion chromatography. Detection limits were in the 0.1-0.5 mg/l range. Schlegel et al. [114] compared direct UV absorption and conductivity detection for arsenic and selenium species. Detection limits were similar (0.4-1.0 pg/ml) but conductivity detection was more universal. Vogt and Werner [115] discussed the speciation of inorganic and organic arsenic and selenium species using CE with indirect UV detection. All four species could be separated using a chromate buffer system at a pH of 10. HTAB was used to reverse the electroosmotic flow and a negative voltage was applied to the inlet end of the capillary. Greschonig et al. [116] described the separation of arsenite, arsenate, dimethylarsinic acid, diphenylarsinic acid, methanearsonic acid, phenyl-aminophnyl arsonic acid, paminopnyl arsonic acid, phenylarsineoxide and phenarsazinic acid. UV absorption detection was used at 200 nm (Fig. 6.9b). The electrolyte buffer was 15 mM phosphate at pH 6.5 and 10 mM sodium dodecylsulfonate. Selenite, selenate, selenocystine and selonomethionine were separated by CE and detected using indirect UV absorption in standards [117] as well as thermal and mineral waters [118]. A chromate electrolyte was used along with TTAB to modify the electroosmotic flow. High concentrations of nitrate (ratio of 1:5 selenite:nitrate) interfered with the detection of selenate. Very high concentrations (>20 mg/1) of carbonate interfered with selenite detection. This could be overcome using element selective detection. Detection limits were about 10 ng/ml for the inorganic selenium species and 300 ng/ml for the organic selenium species. Casoit et al. [119] simultaneously measured arsenic, selenium, antimony and tellurium speciation in waters and soil abstracts using CE with indirect UV absorption detection. Sodium trimethyltetradecylammonium hydroxide (TTAOH) was added to the electrolyte/ buffer in order to reverse the direction of electroosmotic flow and reduce analysis times. After optimization of the pH and electroosmotic flow modifier concentration, simultaneous analysis could be accomplished in 5 min. The Se(VI) peak could not be separated from the carbonate peak when the pH was less than 9. Several peak overlaps inhibited detection of several of the species in drinking water. Hagege et al. [120] described optimization of CE conditions for separation and detection of Se(VI), Se(IV), selenomethionine, and selenocystine. 185

As illustrated in some of the above descriptions, two problems with CE analysis of arsenic and selenium species, as well as other species, are inadequate detection limits and peak overlaps due to other species (in this case other anions present). Potential solutions to these limitations are means to inject more analyte, more sensitive detectors and more highly selective detectors. Large-volume stacking with [34] and without [35] polarity switching can be used to improve detection limits. [35]. Up to 50% of the capillary was hydrodynamically filled with the low conductivity sample [35] and then stacked. Ten to twenty-fold sensitivity (peak height) enhancement for DMA, MMA and arsenic acid was obtained using the large volume stacking with polarity switching. Quantitative results could be obtained if the conductivities of all of the samples were made to be the same using 100x diluted electrolyte/buffer solution. Up to 80% of the capillary was filled with sample solution and then the sample was stacked without polarity switching. In this case the sensitivity enhancement was a factor of 30-40 with less peak broadening than the polarity switching approach. Li and Li [121] reported the use of preconcentration of selenium and arsenic species using field amplification (large volume stacking) for natural water samples. Preconcentration factors over 100 were achieved in less than 4 min leading to detection limits that were less than 25 ng/ml. 6.4.2.2 Arsenic and selenium speciation using element selective detection Element selective detection, using ICP-MS and ICP-OES, has also been used with CE for arsenic speciation. Improved detection limits and higher selectivity that overcomes potential overlaps with other anions should be advantages of these detectors. Liu et al. [61] used a direct injection nebulizer (DIN) based interface to couple CE with ICP-MS. Hexamethonium hydroxide was added to the electrolyte/buffer solution to reverse the direction of electroosmotic flow. A voltage of -30 kV was applied to the inlet end of the capillary while the exit end of the capillary was grounded through a sheath flow solution flowing at 15 1l/min. Detection limits for As(III) and As(V) were 100 and 20 pg/ml, respectively, although the authors did not specify whether hydrodynamic or electromigration injection was used. Concentration based CE-ICP-MS detection limits were similar for As(III) as for continuous sample introduction at 100 pl/min while the CE-ICP-MS detection limits were five 186

times worse for As(V). Most other species had CE-ICP-MS detection limits that were about 15 times higher than for continuous sample introduction which probably indicates that the As species were injected electrostatically in order to obtain some preconcentration. Two groups [122,1231 very recently reported the use of CE-ICP-MS to separate and detect six arsenic species: including anions (arsenite, arsenate, MMA, DMA) and cations (arsenobetaine (AsB) and arsenocholine (AsC)). Two different approaches were used to detect all of the species from a single injection. Michalke and Schramel [122] used a two-step separation then detection process. They injected a 200 mM phosphate buffer solution, then the sample (375 nl), then a 20 mM phosphate solution and acetonitrile. The sample was positioned approximately 10 cm inside of the inlet end of the capillary. Voltage (-18 kV) was applied to the capillary for 12 min. Then buffer/electrolyte solution was forced into the capillary at a laminar flow rate of approximately 1.5 l/min by pressurizing the inlet electrolyte/buffer reservoir. The separated species then moved to the ICP-MS driven by the induced laminar flow. As a result, all of the species could be detected from a single injection (Fig. 6.10). Detection limits were between 15 and 65 ng/ml. However, these were too high to observe As at natural levels in urine. The observed As' signal followed the Cl1 signal, suggesting that the signal at mass 75 was due to AsCl. Arsenic speciation in the liquid phase of a sewage sludge sample was successful with the detection of MMA and As(V). However, another peak due to an arsenic containing species was not identified. Van Holderbeke et al. [123] added Anion-BT to reverse the electroosmotic flow in CE-ICP-MS. The six arsenic species were separated in about 1300 s (Fig. 6.11). The AsC, which is cationic at the pH used, may have been observable because of some residual laminar flow induced by the nebulizer (Fig. 6.11a). By applying pressure to the inlet reservoir after the As(V), MMA, DMA and As(III) were detected, a laminar flow was induced that reduced the AsC migration time from about 1300 to 600 s. Detection limits for the inorganic arsenic, MMA and DMA species were reported to be in the 1-2 ng/ml range, using sample stacking. Concentrations of DMA, AsB and AsC in human urine after a fish dinner were measured to be 29, 83 and 33 ng/ml, respectively. External and spike addition calibrations produced similar results. A peak at the migration time for As(V), corresponding to an anomalously high concentration of 100 ng/ml was observed. It appears that this was due to a spectral overlap due to ArNaC'. 187

AsC, AsB

(n 0o

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40

50

SO

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90

100

110

120

seconds

u)

0

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10

20

30

40

60

60

70

80

S0

100

110

120

seconds

Fig. 6.10. Arsenic speciation using CE with ICP-MS detection and co-electroosmotic flow [122]. Reprinted by permission of John Wiley & Sons, Inc.

Kirlew et al. [124] used an ultrasonic nebulizer based CE-ICP-MS interface to measure arsenate, arsenite and DMA. Detection limits were reported to be 84, 95 and 158 ng/ml for arsenate, arsenite and DMA, respectively, when hydrostatic sample injection was used. Electrokinetic sample injection provided a factor of 25-30 improvement in detection limits. 188

Fig. 6.11. Arsenic speciation using CE-ICP-MS and counter-electroosmotic flow. (a) Electropherogram of six arsenic species a t 20 nglml As using ICP-MS detection [I231 and counter-electroosmotic flow. (b) Electropherogram of four arsenic species a t 20 ng/ml under optimized conditions for CE-ICP-MS [1231.Reproduced by permission of The Royal Society of Chemistry.

Arsenic is monoisotopic and can suffer from spectral overlaps from ArCl', NaCAr+, CPO,', 36Ar38Ar'H+as well as other polyatomic ions, with ArCI' being particularly common in solutions containing high chloride concentrations. One way to reduce the signals due to these polyatomic ions is to use a hydride generation based sample introduction system. Magnuson et al. [76,77,1251 used electrokinetic injection and hydride generation with a gas-liquid membrane separator. Detection limits of 25, 6, 9 and 59 pglml were obtained for arsenite, arsenate, MMA and DMA, respectively. Detection limits for Se(V1)and Se(1V) were 10 and 24 pg, respectively, a n injection volume of 250 nl. These correspond to concentration based detection limits of 40 and 96 nglml. Tian et al. [781 tested a movable reduction bed hydride generation system to couple CE and ICP-OES. Using a relatively large, 400 nl hydrostatic injection volume into a 100 pm i.d. capillary, they obtained detection limits (0.3 pglml) that were about ten times poorer than for continuous sample introduction into ICP-OES. CE-ICP-MS has also been used for selenium speciation [60,61, 126-1291. Se(VI), Se(IV), selenium carrying glutathione, selenometh-

ionine, selenocystine and selenocystamine were separated and detected from a single injection using the two step separation, then detection with forced laminar flow approach described above [128]. Detection limits were 10 to 20 pg Se/l for the inorganic species and 35 to 50 Vg Se/l for the organic species. The technique was applied to human milk and serum [126]. Migration times may be different in standards than samples, so spiking may be necessary to identify species by CE-ICP-MS. Van Holderbeke et al. [123] reported that migration times in volvic mineral water and soil leachate were up to 35 and 50 s shorter, respectively, than in standards. Migration time differences were even larger (up to 150 s) for urine samples. 6.4.2.3 Arsenic and selenium speciation using electrospray mass spectrometry detection Electrospray (ES) or ion spray (IS) mass spectrometry has the potential to unequivocally identify species separated by CE. Corr and Anacleto [130] demonstrated the use of CE-IS-MS of inorganic species, including inorganic arsenic and selenium species. Schramel and Michalke, who have been among the most active researchers in the area of CE with ICP-MS and ES-MS for elemental speciation, recently reported on the use of CE-ES-MS for selenium speciation [131,132]. Selenomethionine, selenocystamine and selenocystine were separated from each other by CE and detected using ESIMS. A previously used CE separation method using a Na 2 CO3-NaOH system could not be used with ESI so a 2% acetic acid solution was used instead. High concentrations of non-volatile species are known to cause problems with electrospray ionization including poor sensitivity, extensive signal fluctuation and sometimes an inability to produce an adequate spray. The authors found that the position of the electrophoresis capillary relative to the metal tip of the electrospray needle needed to be carefully optimized. The identification of the three selenium species could be made from the electrospray mass spectra. However, detection limits were two to three orders of magnitude poorer than those using CE-ICP-MS. 6.4.3 Anion speciation A variety of anions including nitrate, nitrite, bromide, bromate, sulfate, sulfite, carbonate, chloride, chlorate and many different phosphorous 190

TABLE 6.3 Examples of some inorganic anions amenable to speciation by capillary electrophoresis bromate

hexametaphosphate

bromide

hypophosphate

chlorate

phosphate

chloride

phosphite

chlorite

pyrophosphate

hypochlorite

trimetaphosphate

perchlorate

sulfate

iodate

sulfite

iodide

thiosulfate

compounds have been separated and detected using CE. Some of the anions that can be speciated using CE are listed in Table 6.3. The recent review of capillary electrophoresis of inorganic anions by Kaniansky et al. [133] contained 293 references. The advantages of CE compared to ion chromatography (IC) include high efficiencies and peak capacities [5]. Figure 6.12 shows the separation of 17 anion species in less than 2 min using CE. The electrolyte/buffer was 5 mM chromate and 0.4 mM OFM Anion-BT (to reverse the electroosmotic flow). A 60 cm long, 50 gm i.d. fused silica capillary was used. The sample was injected electrostatically using -1 kV for 15 s. The separation voltage was -30 kV. In contrast to IC, the migration order is predictable from equivalent ionic conductance values of the species. CE has been used for speciation of phosphorous containing species [134-136]. Twelve polyphosphates and 12 polyphosphonates were separated using CE with indirect UV absorption detection [137]. Detection limits were 0.8 to 11 pM, comparable to commonly used HPLC methods. This technique was used to measure phosphates and phosphonates in soap and toothpaste. Polyphosphates were measured in detergent using CE [78] with detection limits around 2 x 10 -5 M. Adenosine 5triphosphate was used as the chromophore for indirect UV absorption detection (260 nm). Cetyltrimethylammonium bromide was used to reverse the electroosmotic flow. Halides and oxoanions of halides have also been measured by CE. Shamsi and Danielson separated a group of seven anions [138]. 191

6 78 4 1' a

33

fr 1.40

1.60

1.80

2.00

2.20

2.40

2.60

35

t

34-~3

2.80

Minutes Fig. 6.12. Separation of 18 anion species in less than 2 min. Peaks include: 1: thiosulfate, 2: bromide, 3: chloride, 4: sulfate, 5: nitrite, 6: nitrate, 7: molybdate, 8: azide, 9: tungstate, 10: monofluorophosphate, 11: chlorate, 12: citrate, 13: fluoride, 14: formate, 15: phosphate, 16: phosphite, 17: chlorite, 21: chlorate. Peaks 18-20, 26-36 are small organic anions. A 60 cm long, 50 pm i.d. fused silica capillary was used with a 5 mM chromate, 0.4 mM OFM Anion-BT adjusted to pH 8.0 electrolyte/buffer. The applied voltage with -30 kV. Electrokinetic injection was used at -1 kV for 15 s. From [194].

Indirect UV absorption detection limits were enhanced by up to a factor of five when the electrolyte and analyte mobilities were closely matched. Detection limits were 16 to 300 jig/l. Chloride, chlorate, perchlorate and chlorite were measured in tap water using CE in the presence of nitrate and sulfate [139]. Sulfur speciation is also amenable to capillary electrophoresis as illustrated by Motellier et al. [140]. Chromate electrolytes can oxidize some sulfur species. Benzenetetracarboxylic acid or naphthalenedisulfonate were found to be effective alternatives as indirect UV chromophores to avoid oxidation of sulfur species. The electrolyte/ buffer also contained 10 mM Tris(hydroxymethyl) aminomethane and 0.5 mM diethylenetriamine, which reduced the electroosmotic flow rate. Indirect photometric detection is most commonly used for anion measurements, as described in the recent review by Doble and Haddad [141]. Detection limits are typically in the 0.1-0.5 ,ug/ml range with 192

hydrostatic or hydrodynamic injection and an injection volume of about 40 nl [5]. Linear dynamic range is typically 0.1-100 pg/ml [5]. Electromigration sample injection with isotachophoretic stacking can improve detection limits to the low ng/ml range for some samples [5]. Nutku and Erim used CE to simultaneously measure Br, I-, NO2-, NO3 - and SCNin tap water and human urine [142]. The capillary was coated with a cationic polymer, polyethylene-imine. Conductivity based detection, which relies on differences in conductivity between the analyte peak volume and the electrolyte/buffer solution, can also be used. Large differences in conductivity can lead to band broadening, so some trade-offs are necessary [133]. Some micropotentiometric detectors have been used as detectors for CE of anions

[55]. Because many anions might be present in real samples and each must be separated from all of the rest of the anions when non-selective detection is used, means to affect migration times by complexation [143], chemical parameters [144] or instrumental parameters [144] are often key to successful measurements. 6.4.4 Metal binding with organic ligands and biomolecules Capillary electrophoresis has been widely used for binding studies [145-151] although most of the applications were for protein, peptide and sugar binding to proteins, DNA and peptides. Weakly binding ligands, such as hydroxyisobutyric acid (HIBA), are often used to improve the separation of inorganic cations with similar charge using CE [152-157]. Capillary electrophoresis has the potential to measure the relative concentration of free metal ions and metal-ligand complexes as well as to investigate their kinetics and complexation constants. 6.4.4.1 Effect of complexation on migration times and CE separations When mixtures of free metal ions and complexing ligands are investigated by CE, the binding strength and exchange kinetics must be considered. If the metal is strongly bound to the ligand and the exchange of free metal ions and bound metal ions is slow, separate peaks will be observed in the electropherogram. An example is the separation of Cu 2+ and Cu-EDTA, shown in Fig. 6.13. However, if the exchange kinetics are rapid, only a single peak will be observed. In this case, the observed migration time will depend on the concentration 193

a,

0

E

8

LC

0

50

100

150

200

250

Time (s)

Fig. 6.13. Separation of Cu 2+ and Cu-EDTA. A positive laminar flow was used to detect Cu 2 + and Cu-EDTA2 from a single injection. ICP-MS detection was used. Reprinted with permission from [44]. Copyright (1995) American Chemical Society.

weighted average of the different species, as shown in Fig. 6.14. The complexation constant may be determined from the observed migration time, as discussed below. 6.4.4.2 CE of metal-ligandcomplexes Depending on their thermodynamic and kinetic stabilities, free ligands, metal-ligand complexes and free metals can be separated using CE. Btirgisser and Stone [158] reported speciation of solutions containing Co(II), Co(III), ethylenediaminetetracetic acid (EDTA), nitrilotriacetic acid and other amino carboxylic acids. These are important because EDTA and NTA were often used in the processing of nuclear materials. Co(III)-EDTA complexes are very stable and adsorb poorly to subsurface minerals so that the transport of radioactive 60Co is enhanced [159]. A phosphate electrolyte/buffer was used at pH 7. Trimethylammonium bromide (TTAB) was added to reverse the direction of electroosmotic flow so that the migration complexes and free ligands would be in the same direction as the electroosmotic flow when the capillary inlet was held at a negative potential relative to the exit end of the capillary. The authors also noted that the TTAB prevents metal ions and free amino carboxylic acids from adsorbing to the fused silica capillary walls. Using UV absorption detection, the peak 194

500 400 a 300

100

n 0 500

. CO

200

b

400-

X

100

300

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0.41 pM GA

400 P300

200 +

100

0

0

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0

200

300

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C

3.3 pM GA

400

•

300-

2200 100 0

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100

200

300

400

Migration time (s)

Fig. 6.14. Change in migration time for Co2+-glutaric acid complex as a function of added glutaric acid using CE-ICP-MS detection. (a) No glutaric acid in sample. (b) Glutaric acid present in sample at concentration equivalent to 25 pg/ml C. (c) Glutaric acid present in sample at concentration equivalent to 200 pg/ml C. The pH was 6.0 for each sample. From [167].

area depends on both the concentration and the migration velocity. Therefore, the ratio of peak area to migration time was used for quantitation. CE has been used to separate 4-(2-pyridylazo) resorcinol and its complexes with Co(III), Fe(II), Ni(II) and W using photometric detection [160]. Aguilar et al. [161] used CE to measure metal cyanide complexes of Au, Ag, Cu, Ni and Fe in gold processing solutions. On195

capillary stacking was used to obtain detection limits in the mid part per billion range expressed as the metal. CE has been used to determine metal cyanide complexes in electroplating baths [162] and leaching solutions from automobile catalytic converters [163]. 6.4.4.3 Determinationof metal-ligand complexation constants Erim et al. [164] considered the applicability of CE to study metalligand complexation in solution in 1994. They concluded that CE has attractive features for the measurement of complexation constants but that more sensitive detectors were needed. ICP-MS may provide the necessary sensitivity for many systems. Expressions to estimate association constants from capillary electrophoresis measurements have been described by Rundlett and Armstrong [165]. Erim et al. [164] described three approaches to measuring complexation constants using capillary electrophoresis. They investigated neutral ligands so that the free metal and metal-ligand complexes would have a similar charge and mobility. This would ensure that the equilibrium was maintained during the measurement. In frontal analysis, a large sample plug that contains the buffer, metal and ligand is injected into the capillary. Then a positive voltage is applied. The free elemental ion exits the front edge of the sample plug because it has a higher mobility than the ligand or metal-ligand complex. The free ligand exits the rear end of the sample plug because it has the smallest mobility (and migrates due only to electroosmotic flow if it is neutral). As a result, three plateaus can be observed in the electropherogram. The first is due to the free metal ion, the second is due to the metal ligand complex and the third is due to the free ligand. Figure 6.15a shows the electropherogram for Cu2+-1,10 phenanthroline complexation using direct UV absorption detection. The plateau for Cu 2+ is not observed because the ion does not absorb light at the wavelength used. A second approach is called the Hummel-Dreyer method. In this case, the electrolyte/buffer reservoirs are filled with solution that also includes the ligand. A small sample of electrolyte/buffer and free metal ion are injected. The ion and ligand are mixed in the beginning of the capillary because of their different mobilities. In the injected plug, the ligand concentration is reduced due to complexation with the metal ion. Using UV absorption detection a negative peak is observed that moves with the mobility of the ligand, as seen in Fig. 6.15b. The metal-ligand complex moves in the electrolyte buffer and ligand so the local UV absorption increases, producing a positive peak. 196

A

1

2

0

5

min

Fig. 6.15. Electropherograms from Cu2 +-l,10-phenanthroline complexation measured by CE. (a) Frontal analysis method, electrokinetic injection for 60 s at 10 kV, 20 kV electrophoresis voltage. Plateau 1 is due to the metal-ligand complex. Plateau 2 is due to free ligand. (b) Hummel-Dreyer method, injection for 2.4 s at 10 kV, 12 kV electrophoresis voltage. The first peak is due to the complex. The second peak is due to the deficiency in ligand. (c) Vacancy peak method, injection for 2.4 s at 10 kV, 12 kV electrophoresis voltage. The first peak is due to the deficiency in metal-ligand complex. The second peak is due to the deficiency in ligand. From [164].

The vacancy peak method can also be used. The electrolyte/buffer reservoirs also contain both the ligand and metal. A small injection of electrolyte/buffer solution without the ligand or metal is injected. Voltage is then applied. As the injected plug moves, ligand enters from the leading edge because its mobility is low. At the trailing edge, the metal and metal-ligand complex move into the injected plug because their mobilities are higher. After some time, the metal and metal-ligand intersects the ligand and the complex will reach an 197

15 10

o:3

0.00

0.02

0.04

0.06

0.08

0.10

1/AT (s-') Fig. 6.16. Effect of glutaric acid concentration on Co species containing peak migration -4 time. The pH was 6.0. Concentration of Co in the original sample was 1.0 x 10 M. A Teflon® capillary and nebulizer were used. Source: [167].

equilibrium concentration. Following that time, the deficiencies in free ligand and complex will migrate towards the detector. The resulting electropherogram, detected using direct UV absorption, will have two negative peaks, the first with a deficiency of the metal-ligand complex and the second for the deficiency of the free ligand (Fig. 6.15c). Erim et al. [164] found that the stability constants measured for the Cu(II)-1,0 phenanthroline and Cu(II)-2,2'-bipyridyl complexes measured by each of the three methods using CE were similar to those reported in the literature. Takayangai et al. [166] used CE to monitor complex formation and ion association of alkali metal ions, dibenzo-18crown-6, picrate and perchlorate. Weakly bound ligands with rapid exchange kinetics have been widely used to improve resolution of inorganic cations using CE. The migration time depends on the time weighted average mobilities of the free metal ion and the metal ligand complex. The time-weighted average depends on the extent of complexation. The migration times are predictable based on known mobilities and complexation constants. Therefore, it is also possible to determine complexation constants by measuring migration times for the single peak due to the rapidly exchanging free metal ion and metal-ligand complex. Figure 6.16 shows the change in migration time for Co 2+ and its complex with glutaric acid as a function of glutaric acid concentration [167]. The equilibrium constant for one to one metal-ligand binding can be calculated from the following relationship: L

[L] 198

( E't)

K

where [L] is the ligand concentration, K is the complexation constant, Atm,, is the difference in migration times for the metal-ligand complex and the free metal ion, and At is the difference in the migration time for the observed peak. The value ofK at pH 6 was determined from the intercept of Fig. 6.16. The experimentally measured value for log K was 2.48 while the literature reported value was 2.28 [168]. Knowing the value of K, the experimentally measured value for Atm,, can be calculated from the slope. Capillary electrophoresis can be used to determine complexation constants for chemical systems with formation rates that are too slow for potentiometric determination [169]. For example, complexation constants for a series of lanthanide(III) elements with 1,7 dicarboxymethyl-1,4,7,10-tetraazacyclododecane were measured using CE [169]. The free ligand and metal-ligand complexes were determined. Samples prepared by mixing metal ions and ligands were allowed to equilibrate 12 h before measurement. The pH was adjusted so that about 50% of the metal was complexed but metal hydroxides did not precipitate. A 25 cm long, 50 pm i.d. uncoated fused silica capillary was used at voltages between 8 and 15 kV. Peak heights were measured. Using the initial concentrations of ligand and metal ion and the concentrations of free ligand and metal complexes, a conditional stability constant at a specific pH can be calculated. Then the stability constant of the metal-ligand complex can be calculated. 6.4.4.4 Metal complexation with humic and other polyhydroxy substances The distribution of trace metals in natural waters may be highly dependent on the extent of binding to humic and fulvic substances. However, this metal complexation can be difficult to measure reliably. Depending on their source, humic substances can have molecular weights from a few hundred to several hundred thousand. Capillary electrophoresis may offer unique capabilities to investigate metalhumic substance interactions. Challenges include minimizing metal and humic substance interactions with the walls of the capillary and properly selecting buffers to control pH without interacting with the species of interest [170,171]. Attempts to 'optimize' separations to obtain only 'sharp peaks' may lead to changes in speciation if great care is not exercised. Norden and Dabek-Zlotorzynska [57] recently reported investigations of metal-fulvic acid interactions using CE with uncoated fused 199

silica capillaries. Two different approaches were used. In one, the concentration of free metal ions was measured with and without fulvic acid in the sample. The decrease in the peak area of the free metal ion was used to determine the extent of complexation. Because UV absorption detection was used, CDTA was added to the electrolyte/buffer in order to form a detectable metal-CDTA complex. However, the complexation of the metal ions with CDTA could compete with complexation with the fulvic acid and lead to apparently smaller metal-fulvic acid complexation. The second approach focused on the migration behavior of the fulvic acid in the absence and presence of Fe 3+ or Hg 2+. Changes in peak shape and area were observed for samples containing the metals that were consistent with metal-fulvic acid complexation. However, quantitative interpretation was hampered by the use of a non-specific detector and potential fulvic acid-capillary wall interactions. The order of apparent extent of complexation obtained using the first approach (Al3+ > Hg 2+ > Cu 2+ > Pb2 + > Sr 2 +) was consistent with previously reported results using other methods. These researchers also reported the use of CE with UV absorption and laser-induced fluorescence detection for the characterization of an aquatic fulvic acid and a solid humic acid. Changes in the electropherograms when Al3+ was present in the sample indicates that there are specific binding sites for Al3+. Keuth et al. [172] used free flow isotachophoresis to separate humic acid fractions and then CE to assess individual fractions. Two sharp peaks were observed for pure humic acid at pH 12. CE with a coated capillary has also been used to measure metal speciation in plant and food extracts [173]. 6.4.4.5 Metal binding to biological molecules Capillary electrophoresis has been widely used to separate biological molecules. Therefore, it is natural that CE has also been used for analysis of metalloproteins and metal binding with other macromolecules [174]. Metallothioneins are involved in homeostatic control, metabolism and detoxification of several trace metals. Beattie [175] recently reviewed strategies for analysis of metallothioneins by CE. Small differences in the chemical properties (charge or hydrophobicity) of metallothionein isoforms leads to the need for separation techniques that can be optimized to provide high resolution. Beattie [175] suggested that CE was the ideal technique for this purpose. Among the challenges of metallothionein analysis is achieving necessary sensi200

tivity for real samples. Preconcentration by sample stacking or solid phase extraction [175] or use of detectors with high sensitivity (such as ICP-MS) are two means to improve detection limits. Lobinski et al. [176] reviewed the use of element selective detections with size exclusion chromatography, reverse-phase HPLC and CE for metallothionein analysis. Uncoated capillaries and capillaries with a variety of coatings have been assessed for metallothionein separations. Beattie [175] concluded that qualitative measurements are best made using low pH phosphate buffers with a polyacrylamide coated capillary (neutral surface). Quantitative measurements were best with uncoated fused silica capillaries and borate-SDS electrolyte/buffer solutions at alkaline pH. Many of the reports focusing on interfaces between CE and ICP-MS included an assessment of electrophoretic resolution and detection limits for metallothioneins [59,62-64,74]. Detection limits in the 2-10 fg range have been reported with concentration based detection limits as low as 0.05 ng/ml. Wittrisch et al. [177] used proton-induced X-ray emission (PIXE) to detect copper, zinc and cadmium containing metallothionein isoforms. Electrospray and ion spray MS would seem to provide an ideal detector for CE of metallothioneins if detection limits are sufficiently low. Guo et al. [178] very recently reported results using CE-ESI-MS for the analysis of metallothioneins. Detection limits were estimated to be approximately 0.6 fmol per sub-isoform. Metal-peptide binding has also been investigated using CE [179, 180]. Changes in mobilities of the proteins were used to verify specific metal binding. The binding of metals to aminoacids and citrate has been monitored using CE [181]. Semenova et al. [182] investigated the stability of zinc complexes with caffeine, nicotinic acid, thiourea and phenazone using CE. For the thiourea and phenazone complexes degradation products including the free zinc ion, carboxylate ligand and the neutral ligand were observed within a few hours in water. Zhao et al. [183] investigated various forms of ferritins. O'Keefe et al. [184] used CE to study metal-polyaminopolycarboxylate complexes and effects of metals on proteins. Metal binding to pentadecapeptide from Panax ginseng has also been assessed [179,180]. Metal-DNA interactions have been observed using CE [185]. The degradation of s86Re-l-hydroxyethylidene complex, that is used for radiotherapeutic treatment of humans, has been monitored using CE [177]. 201

6.4.5 Other applications CE has been used for a variety of other elemental speciation measurements. Organotin compounds can be separated well by CE [173, 186-188]. However, indirect UV absorption or fluorescence detection limits are often in the part per million range which is insufficient for routine environmental analysis. ICP-MS detection should be capable of providing the part per billion detection limits that are needed for environmental applications. Mercury speciation of inorganic mercury and organomercury compounds can be achieved within 12 min using CE [189,190]. Using amperometric detection and electrokinetic injection, detection limits in the range 0.2-5 lig/l can be achieved [6]. CE separation of inorganic ions might also be used to avoid isobaric overlaps in ICP-MS, particularly when the sample volume is limited or when handling only very small sample volumes is advantageous, such as with radioactive samples. For example, measurement of long-lived radioisotopes is complicated by isobaric overlaps [191] such as 107Pd+ and 107 Ag+, 15 1Sm+ and 15 1Eu+, 93Mo+ and 93Zr+ and 93Nb+, 99Tc+ and 99Ru+, 12 6 Sn+ and 126 Te+ and 126 Xe+. Sutton et al. [192] have reported the CEICP-MS measurement using hydroxylisobutyric acid (HIBA) as an element dependent, weakly complexing ligand [155] in order to separate lanthanides. Inorganic polymerization is important in environmental systems, some catalysts and products (antiperspirants, for example). Iron, aluminum, zirconium and chromium compounds can be formed by hydrolytic polymerization. As polymerization proceeds more highly charged species are produced. For example, chromium species that might be produced include Cr2 (OH)24+, Cr 3(OH)4 5+, etc. The polymerization process is slow and highly pH dependent. Figure 6.17 shows electropherograms obtained from solutions that were aged 30 days at various pH values [193]. The CE analyses show that more highly mobile (and therefore, more highly charged) species are formed as the pH is increased from 3.6 to 4.3. While ICP-MS detection provided element specificity and the migration behavior is related to the charge on each species, the molecular identity of each species was not obtained directly. Electrospray or ion spray MS could provide more direct identification. IS-MS spectra measured of the mixtures without CE separation showed that polymeric species with up to +5 charge were present. 202

200

150

v,

CO

0

o 50 0

200

300

400

500

600

Migration time (s) Fig. 6.17. Electropherograms of aged solutions of CrCI 3 at various pH values. Source: [193].

6.5 CONCLUSIONS: ADVANTAGES AND LIMITATIONS OF CAPILLARY ELECTROPHORESIS FOR ELEMENTAL SPECIATION The advantages of CE include small sample volumes, high efficiency and the ability to use a wide variety of electrolyte/buffer solutions so that specific speciation of interest can be maintained. The physical rather than chemical partitioning based separation basis may aid in preventing some types of changes in speciation during separation. Resolution-analysis time tradeoffs can be easily made. CE can be used to separate anions, cations and neutral species. The disadvantages of CE for elemental speciation include the small injection volumes that can lead to degradation of concentration based detection limits, potential loss of analyte ions in the capillary, potential changes in speciation during separation and deleterious sample-wall interactions. CE sample-wall interactions can be particularly problematic. For example, the Co2+-glutaric acid complexation constant could not be measured using a bare fused silica capillary because the addition of glutaric acid had no effect on the observed peak migration time. However, CE using a Teflon® capillary was successful for this same chemical system. 203

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