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MICELLAR ELECTROKINETIC CHROMATOGRAPHY
MICELLAR ELECTROKINETIC CHROMATOGRAPHY
1
MICELLAR ELECTROKINETIC CHROMATOGRAPHY S Terabe and J-B Kim, University of Hyogo, Hyogo, Japan & 2005, Elsevier Ltd. All Rights Reserved.
Introduction Over the last two decades, capillary electrophoresis (CE) has been developed as a powerful separation technique for complex mixtures. Its advantages include a high separation efficiency, short analysis time, small sample requirement, and applicability to a wide range of analytes. The basic modes of CE that are presently being exploited include capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), capillary isotachophoresis, capillary isoelectric focusing, and capillary gel electrophoresis. In CZE, only ionic or charged analytes can be separated in principle since its separation mechanism is based on the difference in electrophoretic mobility of the analytes. MEKC has become popular as a powerful technique for the separation not only of neutral analytes but also of charged ones using a conventional CE instrument without any alteration. In MEKC, an ionic surfactant micelle is used as a pseudostationary phase (PS) that corresponds to the stationary phase in conventional chromatography and the surrounding aqueous phase to the mobile phase. The separation principle of MEKC is based on differential partitioning of analytes between the aqueous phase and the micelle phase. MEKC is a mode of electrokinetic chromatography (EKC) in which surfactants (micelles) are added to the buffer solution. They have polar head groups that can be cationic, anionic, neutral, or zwitterionic and nonpolar hydrocarbon tails. The formation of micelles is a direct consequence of the hydrophobic effect. The surfactant molecules can self-aggregate if the surfactant concentration exceeds a critical micelle concentration (CMC). The hydrocarbon tails will then be oriented toward the center of the aggregated molecules, whereas the polar head groups point outward. Micellar solutions can solubilize hydrophobic compounds that would otherwise be insoluble in water. Every surfactant has a characteristic CMC and aggregation number (AN), i.e., the number of surfactant molecules making up a micelle (typically in the range of 50–100) (see also Table 1). The size of the
micelles is in the range of 3–6 nm in diameter; therefore, micellar solutions exhibit properties of homogeneous solutions. Micelles have a dynamic structure that is the result of the rapid equilibrium between aggregated and monomeric forms. It is the differences in interaction between the micelle and the neutral solute that cause the separation. Micellar solutions have been employed in a variety of separations and spectroscopic techniques.
Micelles as Pseudostationary Phase for MEKC The CMCs and ANs of various kinds of surfactants suitable for PSs in MEKC are listed in Table 1. An anionic surfactant, sodium dodecyl sulfate (SDS), is most widely used as a PS in MEKC. Its popularity can be attributed to its low CMC, high aqueous solubility, low Krafft point, ready availability and the low cost of the pure product. Cationic surfactants such as tetradecyltrimethylammonium bromide (TTAB), cetyltrimethylammonium bromide (CTAB), and cetyltrimethylammonium chloride (CTAC) have also been useful for MEKC analysis. Most cationic surfactants have an alkyltrimethylammonium group, and their counterions are halides. The addition of cationic surfactants to the background electrolytes (BGE) caused the reversal of electroosmotic flow (EOF) owing to a positively charged capillary wall on account of the adsorption of cationic surfactants. As a result of the reversed EOF, the polarity of the electrodes has to be reversed in order to detect the analytes. Nonionic surfactants themselves do not possess electrophoretic mobility, but they have the distinct advantage of not contributing appreciably to Joule heating; hence, they may be used at high concentrations. They can have a great influence on the separation of charged analytes and can also be employed as PSs in mixed micelles with ionic surfactants. Nonionic surfactants such as polyoxyethylene (23) dodecyl ether (Brij 35), and polyoxyethylene (20) sorbitan monolaurate (Tween 20) have been effectively used as PSs for the separation of charged compounds through MEKC. Zwitterionic surfactants are not widely used in MEKC. However, zwitterionic surfactants will be interesting if they are used in mixed micelles or as a modifier of the micelle because they should show
2 MICELLAR ELECTROKINETIC CHROMATOGRAPHY Table 1 CMCs and ANs of selected surfactants
a
Surfactant
CMC a (10 3 mol l 1)
AN
Anionic SDS Sodium tetradecyl sulfate Sodium decanesulfonate Sodium N-lauroyl-N-methyltaurate Sodium polyoxyethylene dodecyl ether sulfate Sodium N-dodecanoyl-L-valinate Sodium cholate Sodium deoxycholate Sodium taurocholate Sodium taurodeoxycholate Potassium perfluoroheptanoate
8.1 2.1 (501C) 40 8.7 2.8 5.7 (401C) 13–15 4–6 10–15 2–6 28
62 138 40 – 66 – 2–4 4–10 5 – –
Cationic TTAB Dodecyltrimethylammonium bromide CTAB CTAC
3.5 15 0.92 1.3
75 56 61 –
Nonionic Polyoxyethylene(23) dodecyl ether (Brij 35) Polyoxyethylene(20) sorbitan monolaurate (Tween 20)
0.1 0.059
– –
Zwitterionic 3-((3-cholamiddopropyl)dimethylammonio)-1-propanesulfonate N-dodecyl-N,N-dimethylammonio-3-propanesulfonate
4.2–6.3 3.3
10 –
251C.
significantly different selectivities from other types of surfactants. Macromolecular surfactants or high-molecularmass surfactants (HMMS), butyl acrylate–butyl methacrylate–methacrylic acid copolymers (BMMAs), and sodium 10-undecylenate (SUA) oligomer have been introduced as PSs for MEKC. Since a HMMS forms a molecular micelle, which consists of one molecule, and the CMC value is essentially zero, one can expect a higher reproducibility in a HMMS– MEKC system compared with a low molecular mass surfactant (LMMS)–MEKC system. Bile salts are biological surfactants synthesized in the liver. The bile salts have both a hydrophilic and a hydrophobic face and tend to combine together at the hydrophobic face in an aqueous phase. To improve selectivity, various types of bile salts have been used as PSs in MEKC. Figure 1 shows the separation and determination of the ingredients of a cold medicine through MEKC with bile salt. Although they are not micelles, microemulsions and cyclodextrin (CD) derivatives have also been introduced as PSs for MEKC. Oil-in-water (o/w) microemulsions have been shown to be good PSs for EKC. Microemulsions (o/w) are prepared by mixing oil, water, a surfactant, and a cosurfactant such as a medium alkyl-chain alcohol. They have the characteristic properties of a solvent, such as thermodynamic stability and a high solubilization power. The
structure of the o/w microemulsion is similar to that of the micelle, except that the microemulsion has a core of a minute droplet of oil. The surfactant and the cosurfactant are located on the surface of the oil droplet to stabilize it. The separation basis of microemulsion EKC (MEEKC) is similar to that involved in MEKC. CD has been widely used not only in liquid chromatography but also in CE as a mobile phase modifier. The use of CD is particularly effective for the separation of aromatic isomers and enantiomers.
Fundamentals Separation Principle
Figure 2 illustrates a schematic representation of the separation principle of MEKC. When an anionic surfactant such as SDS is employed, the micelle migrates toward the anode by electrophoresis. The EOF transports the bulk solution toward the cathode due to the negative charge on the surface of fused silica. The EOF is usually stronger than the electrophoretic migration of the micelle under neutral or alkaline conditions, and therefore, the anionic micelle also travels toward the cathode at a retarded velocity. When a neutral analyte is injected into the micelle solution, a fraction of it is incorporated into the micelle and it migrates at the velocity of the micelle.
MICELLAR ELECTROKINETIC CHROMATOGRAPHY
3
6
8
2 7 10 + 11 14
9
3
5 1
4 12 13
0
5
10
15
20
25
Time (min) Figure 1 Separation of 14 ingredients through MEKC using bile salt. Buffer, 0.02 mol l 1 phosphate–borate (pH 9.0) containing 0.05 mol l 1 sodium deoxycholate; applied voltage, þ 20 V; temperature, ambient; detection wavelength, 210 nm. Solutes: 1, caffeine; 2, acetaminophen; 3, sulpyrin; 4, trimetoquinol hydrochloride; 5, guaifenesin; 6, naproxen; 7, ethenzamide; 8, phenacetin; 9, isopropylantipyrine; 10, noscapine; 11, chlorpheniramine maleate; 12, tipepidine hibenzate; 13, dibucaine hydrochloride; 14, triprolidine hydrochloride. (Reprinted with permission from Nishi H, Fukuyama T, and Terabe S (1990) Journal of Chromatography A 498: 313–323. & Elsevier.)
Detector
: Surfactant
: Electroosmotic flow (EOF)
: Solute
: Electrophoresis of micelles
Figure 2 Schematic illustration of the separation principle of MEKC.
4 MICELLAR ELECTROKINETIC CHROMATOGRAPHY Micelle
Analyte
Water
Capillary Injection (A)
Detector
Water
0
t0
Analyte
Micelle
tR
t mc
Migration time
(B)
Figure 3 Schematic diagram of the zone separation in MEKC (A) and chromatogram (B).
The remaining fraction of the analyte migrates at the velocity of the EOF, and thus the migration velocity of the analyte depends upon the distribution coefficient between the micellar and the nonmicellar (aqueous) phases. The greater the percentage of analyte that is distributed into the micelle, the slower it migrates. The analyte must migrate at a velocity between the electroosmotic velocity and the velocity of the micelle (see Figure 3), provided the analyte is electrically neutral. In other words, the migration time of the analyte, tR, is limited between the migration time of the bulk solution, t0, and that of the micelle, tmc. This is often referred to in the literature as the migration time window in MEKC.
eqns [4] and [5]: k¼
tR ¼ ð1 þ kÞ t0
k¼
nmc naq
½1
We can obtain the relation between the retention factor and migration time for an electrically neutral analyte as k¼
tR t0 t0 ð1 ðtR =tmc ÞÞ
½2
It can be rewritten as tR ¼
1þk t0 1 þ ðt0 =tmc Þ k
½3
When the migration time of the micelle is infinite or the micelle does not migrate in the capillary at all, t0/tmc will be zero; then eqns [2] and [3] become identical to those for conventional chromatography,
½4 ½5
In conventional chromatography, k ¼ N means that the solute is totally retained in the stationary phase and is not eluted at all or the retention time becomes infinite, whereas in MEKC k ¼ N means that the migration time of the solute, tR, is equal to tmc. In that case, such a solute migrates at the same velocity as the micelle or at the lowest velocity and is then detected last. When t0 ¼ 0 or the EOF is completely suppressed, eqn [3] becomes
Retention Factor
The retention factor, k, is defined as the ratio of the number of analytes incorporated into the micelle, nmc, and that in the surrounding aqueous solution, naq:
tR t0 t0
tR ¼
1 1þ tmc k
½6
In this case, the surrounding aqueous phase remains stationary in the capillary and the micelle migrates toward the anode by electrophoresis. Calculation of the retention factor using eqn [2] requires knowledge of t0, tR, and tmc. For the EOF marker of t0, methanol is often used since the distribution coefficient for methanol between the micelle and aqueous phases is negligibly small. Although methanol is UV transparent, it is detectable through the baseline disturbance of the UV detector due to the change in refractive index. Sudan III and Sudan IV are widely used as micelle markers since they are highly lipophilic and totally incorporated into micelle. Timepidium bromide, Yellow OB, and Orange OT are also useful as tracers of micelles. Note that eqn [3] was derived for the retention characteristics of neutral compounds. Hence, relationships between the migration time or mobility and retention factor will be more complicated when the
MICELLAR ELECTROKINETIC CHROMATOGRAPHY
solute has an electrophoretic mobility; that is, the migration time of the ionic solute includes a portion ascribed to the migration of the micelle when the solute is incorporated into the micelle and also another portion ascribed to the electrophoresis of the solute itself. Resolution
The resolution (Rs) in MEKC is given by eqn [7] Rs ¼
pffiffiffiffiffi N a1 k2 1 ðt0 =tmc Þ 4 1 þ k2 a 1 þ ðt0 =tmc Þ k1
½7
where N is the theoretical plate number, a is the separation factor equal to k2/k1, and k1 and k2 are the retention factors of two closely eluting analytes, 1 and 2, respectively. The resolution increases in proportion to the square root of the plate number. The higher the applied voltage, the higher the plate number, unless the conditions are such that the applied voltage generates excessive Joule heating. The average plate numbers for most analytes are usually in the range of 100 000–200 000. If the plate number is considerably lower, analytes are likely to be adsorbed on the capillary wall. In such cases, experimental conditions must be optimized to produce more efficient separations. Cleaning of the capillary is a possible procedure, as is changing the pH of the run buffer. Hydrophobic analytes, or those having longer migration times, typically yield high theoretical plate numbers because the micelle has a smaller diffusion coefficient. This is explained by assuming the major band broadening is due to the longitudinal diffusion as in CZE. The plate number does not depend significantly on the capillary length. With short capillaries, however, the amount of sample volume injected must be minimized to avoid zone broadening. The separation factor, a, is the most important and effective term for maximizing the resolution. The separation factor reveals the relative difference of the distribution coefficient between the two analytes and can be manipulated by chemical means. Since the distribution coefficient is a characteristic of a given separation system, the selectivity can be manipulated by changing either the type of micelle or by modifying the aqueous phase. The optimum value of the retention factor, kopt, for maximum Rs can be calculated by differentiating the following equation, provided N is independent of k: f ðkÞ ¼
k2 1 þ k2
1 ðt0 =tmc Þ 1 þ ðt0 =tmc Þ k1
½8
Then, kopt ¼
rffiffiffiffiffiffiffi tmc t0
5
½9
We can find that kopt is a function of tmc/t0. Under neutral conditions, the optimum value is B2 for SDS micelles as the PS. For practical use, the range of k recommended is between 1 and 5, or at maximum between 0.5 and 10. Unlike conventional chromatography, in MEKC the micelles as the PS migrate with the bulk liquid due to the EOF, in addition to the electrophoretic migration. This migration of the micelle through the EOF does not affect the separation at all. However, the distance of the migration of the micelle cannot be used for the separation. Since the time available for the solute to interact with the moving micelle depends on its k, the column availability depends on k as shown by the last term of the right-hand side of eqn [7] or [8].
Using Modifiers in MEKC MEKC is usually performed with a BGE that consists of just a buffer and a surfactant. Other substances may be added to the BGE to alter the selectivity. Modifiers may affect the charge on the micelle or the solute and change the distribution of the solute between the micelles and the surrounding aqueous phase by interacting with the solute. The following categories of additives are applicable in MEKC: CDs, ion-pair reagents, organic solvents, and others. Cyclodextrins
CDs are oligosaccharides with characteristic toroidal molecular shapes. Their outside surfaces are hydrophilic, while their cavities are hydrophobic. CDs tend to include molecules, which fit their cavities by hydrophobic interaction. The most widely used CDs consist of six (a-CD), seven (b-CD), or eight (g-CD) glucopyranose units. The size of the cavity differs significantly among the a-, b-, and g-CDs. Selectivity results from the inclusion of a portion of a hydrophobic solute into the cavity. The use of CDs is especially effective for the separation of aromatic isomers and aromatic enantiomers that have the chiral center close to the aromatic ring. Many CD derivatives have been developed for increased solubility in water as well as for modifying the cavity shape that provides different selectivities from underivatized CDs and are commercially available. In CD-modified MEKC (CD-MEKC), separation is achieved because of differences in the distribution of the solute among the micelle, CD, and the aqueous phase. The analyte molecule included by CD migrates at the same velocity as the EOF because
6 MICELLAR ELECTROKINETIC CHROMATOGRAPHY
CD electrophoretically behaves as the bulk aqueous phase. The neutral analyte molecule migrates at the same velocity as the EOF, whether included with CD or free from CD. On the other hand, the analyte migrates at a different velocity from that of the EOF when it is incorporated into the ionic micelle. Therefore, the addition of CD reduces the apparent distribution coefficient or k and enables the separation of highly hydrophobic analytes, which otherwise would be almost totally incorporated into the micelle. The higher the concentration of CD, the smaller the distribution coefficient. In CD-MEKC, therefore, the retention factor can be manipulated by varying either the concentrations of the micelle or CD. Enhancement of the separation selectivity of a group of polycyclic aromatic hydrocarbons (PAHs) using CD-MEKC using mixed CDs is shown in Figure 4. Ion-Pair Reagents
Ion-pair reagents are also very useful for selectivity enhancement of both ionic and nonionic analytes in MEKC. For example, when a tetraalkylammonium salt is added to an SDS micellar solution, the migration times of anionic analytes increase with an increase in the concentration of the ammonium salt
because the ammonium ion interacts with the anionic analyte to form a paired ion. Hence, the electrostatic repulsion between the anionic SDS micelle and the anionic analytes is reduced. On the other hand, a cationic analyte competes with the ammonium ion in interacting with the anionic SDS micelle, and so the migration times of cationic analytes decrease with an increase in the ammonium salt concentration. The effect of the ion-pair reagent on selectivity depends strongly on the structure of the reagent, e.g., the length of the alkyl chain. Organic Solvents
Organic solvents miscible with water are widely used as mobile phase modifiers in reversed phase high-performance liquid chromatography (RPLC). Organic solvents may also contribute to the enhancement of resolution or alteration of selectivity in MEKC. However, it should be noted that a high concentration of the organic solvent might break down the micellar structure. In general, the maximum content of the organic solvent is 20–30%. Methanol, 2-propanol, and acetonitrile are widely used as organic solvents in MEKC. The addition of organic solvents usually reduces the EOF and, hence, expands the migration time window. The retention factor is also reduced because the solubility of the analyte into the aqueous phase increases. Other Additives
13 12
1
10 7 9 11 14 2
5 6 3
8
18 1516 17
19 20
4 0
27 Time (min)
54
Figure 4 Separation of 20 PAHs using mixed CD-MEKC. Conditions: Capillary, 65 cm 50 mm inner diameter (50 cm length to the detector); injection by pressure, 0.02 min at 20 mbar, the concentration of each PAH in the mixture was 0.1 mg/ml; UV detection at 230 nm; separation solution, 0.042 mol l 1 b-CD, 0.026 g-CD, 2.5 mol l 1 urea, and 0.100 mol l 1 SDS in 0.140 mol l 1 borate buffer (pH 9); applied voltage, 15 kV. Solute identification: 1, acenaphthene; 2, acenaphthylene; 3, naphthalene; 4, benzo[g,h,i]perylene; 5, fluorene; 6, phenanthrene; 7, pyrene; 8, chrysene; 9, perylene; 10, anthracene; 11, benzo[e]pyrene; 12, benzo[a]pyrene; 13, benzo[a]anthracene; 14, fluoranthene; 15, dibenzo[a,h]pyrene; 16, benzo[k]fluoranthene; 17, triphenylene; 18, benzo[j]fluoranthene; 19, benzo[b]fluoranthene; 20, indeno[1,2,3-cd]pyren. (Reprinted with permission from Jime´nez B, Patterson DG, Grainger J, et al. (1997) Journal of Chromatography A 792: 411–418. & Elsevier.)
A high concentration of urea is known to increase the solubility of the hydrophobic compounds in water. Urea also breaks down hydrogen-bond formation in the aqueous phase. In MEKC, urea slightly reduces the electroosmotic velocity and considerably increases the electrophoretic velocity of the micelle, resulting in an expanded migration time window. The addition of urea is also effective in improving peak shapes, especially in the separation of amino acid derivatives. The selectivity is not remarkably changed by the addition of urea, but minor changes are noticeable, especially for the separation of closely related analytes. Another approach for changing selectivity in MEKC is the use of metal ions. In particular, the MEKC separation of oligonucleotides is improved by the addition of Mg(II), Cu(II), and Zn(II) ions. Metal ions are electrostatically attracted to the surface of a negatively charged micelle where they can be selectively complexed with analytes. Separation is due to differences in the distribution of solutes between the buffer and the metal–micelle surfaces. Retention is proportional to the complexation constant
MICELLAR ELECTROKINETIC CHROMATOGRAPHY
of the oligonucleotide and the metal–micelle surface. Using different metal ions can change the selectivity.
Improving Detection Sensitivity The most widely used detector in MEKC, which is common to other CE modes, is an ultraviolet (UV) photometric detector because many solutes have absorb UV, and the UV detector is easily set up and is cost efficient. One of the drawbacks of UV detection in MEKC is the low concentration sensitivity resulting from a short optical path length, equal to the capillary diameter, and the small injection volumes needed to maintain high efficiency. This limits the applicability of MEKC to trace analysis. Thus, method development is indispensable for reducing the limit of detection (LOD) or increasing the concentration sensitivity. There are different approaches that have been reported for improving the concentration sensitivity in MEKC. These investigations involve the use of highly sensitive detection methods, the installation of capillaries equipped with extended detection path lengths, and offline, and online sample preconcentration methods. The frequently used highly sensitive detection method in MEKC is laser-induced fluorescence (LIF). LODs for this detection mode are at least an order of magnitude lower than that for UV detection. However, in an ordinary laboratory these detectors may not be affordable and UV detection is widely used as previously mentioned. From Beer’s law, the absorbance is proportional to the path length, which is equal to the inner diameter of the capillary. Sensitivity can thus be enhanced through the use of larger inner diameter capillaries. However, this is not a good approach because large-diameter capillaries produce high currents, which cause Joule heating. The separation efficiency deteriorates dramatically when the capillary inner diameter is increased much above 100 mm. Because of problems with large inner diameter capillaries, most approaches to improving the sensitivity have been directed toward extending the path length without increasing the inner diameter of the entire capillary. Bubble cell, Z-shaped cell, and multireflection cell detector configurations have been introduced, which have afforded from twofold to more than 10-fold enhancements in detector response. As with HPLC, a successful MEKC analysis would often rely on dedicated offline sample preparation methods (e.g., liquid–liquid and/or solid-phase extraction). Sample preparation methods usually increase the concentration of the analyte before injection into the capillary. However, these methods require somewhat complex or expensive hardware, with limited applicability, or are time consuming.
7
On the other hand, online sample preconcentration methods based on electrokinetic focusing of large sample volumes represent one of the most facile ways of sensitivity enhancement in MEKC since the preconcentration step is performed within the same capillary used for separation. Two of the most widely used techniques are sample stacking and sweeping. In general, the application of online preconcentration techniques in MEKC is dictated by the specific properties of the analyte and the sample matrix conditions. Thereafter, a variety of online sample concentration techniques have been reported in CZE. They are field-enhanced sample stacking, largevolume sample stacking, transient-isotachophoresis (ITP), pH-mediated stacking, and dynamic pH junction. Sample Stacking
The sample stacking technique was first introduced for ionic analytes in CZE. The basic model of sample stacking is illustrated in Figure 5. In CZE (Figure 5A), the sample is dissolved in a solution that has a lower conductivity than that of the BGE and injected as a longer plug than in normal injection. When a separation voltage is applied, the electric field strength experienced in the sample zone is higher than in the rest of the capillary. The sample ions in the sample zone will move quickly and then slow down when they reach the BGE zone because they experience a lower electric field strength. Consequently, the ions are focused at the boundary of the two zones. It should be noted that the EOF is assumed to be zero in Figure 5A. However, this technique cannot be applied to neutral analytes because neutral analytes have no electrophoretic mobility. In MEKC (Figure 5B), to give effective electrophoretic mobilities to neutral analytes, charged PSs (e.g., anionic SDS micelles) are employed. The sample solution is prepared by dissolving the neutral analytes in a low-conductivity solution and injected into the capillary, which has been previously conditioned with an anionic SDS micellar BGE at neutral pH. The neutral analytes in the sample solution can be quickly carried to the boundary between the BGE and sample solution by the fast migrating anionic micelle entering the sample solution from the cathodic end. Since the electric field strength in the BGE zone is low, the velocity of the micelles is retarded, and the analytes are focused at the boundary between the BGE and the anodic end of the sample solution zone. Note that the neutral analytes are brought to the detector by the EOF since the magnitude of the EOF is greater than the electrophoretic velocity of the micelle.
8 MICELLAR ELECTROKINETIC CHROMATOGRAPHY Stacking boundary (SB) CZE Sample zone
BGS zone High− conductivity
Low−conductivity
Detector
BGS zone High− conductivity
(A) SB EOF MEKC Micellar BGS zone
Electrophoresis of micelles
N
N
Sample zone
N N
Micellar BGS zone
N N
(B)
E
Electric field strength
Figure 5 Schematic diagram of the principle of sample stacking in CZE (A) and MEKC (B).
However, a dispersive effect brought by the mismatch of local EOF velocities between the high and low electric field zones limits the injection length. To solve this problem, the stacking with reverse migrating micelles (SRMM) technique was developed. SRMM employs an acidic micellar BGE to reduce the EOF. Under this condition, the electrophoretic velocity of SDS micelles is higher than that of the EOF. Samples prepared in purified water or in low conductivity matrix are injected for a much longer time compared to the normal injection after conditioning the capillary with micellar BGE. Sample solutions are introduced at the cathodic end of the capillary, and then the separation voltage is applied with negative polarity at the injection end. Since the negative polarity is applied at the inlet, the sample matrix was slowly pushed out of the capillary by the weak EOF. Sweeping
The sweeping technique is defined as a phenomenon where analytes are picked up and concentrated by the micelle, which penetrates the sample zone devoid of micelle. It is independent of the EOF and effective for both charged and uncharged solutes. The principle of sweeping under the acidic condition is schematically shown in Figure 6. In step A, test analytes prepared in a matrix, with conductivity similar to that of BGE but devoid of PSs, are pressureinjected into the capillary at the cathodic end. In step
B, once the separation voltage is applied at the negative polarity with the BGE in the inlet vial, the anionic PS will enter the capillary and sweep the analytes. In step C, the analytes are completely swept by the PS and followed by MEKC separation in the reverse migration mode. Under the optimized condition, B5000-fold enhancements in detection sensitivity were obtained in terms of peak heights by sweeping. Cation- and anion-selective exhaustive injection sweeping (CSEI-sweep and ASEI-sweep) techniques are also available in MEKC. CSEI-sweep and ASEIsweep are combinations of two online preconcentration techniques, sample stacking with electrokinetic injection and sweeping, that can provide more than 100,000-fold enhancements in detection sensitivity. An electropherograms of tap water analysis by CSEIsweep-MEKC is illustrated in Figure 7. The results of this study suggest that CSEI-sweep-MEKC can be used for the analysis of quaternary ammonium herbicides in drinking water.
MEKC Hyphenation Technique with Mass Spectrometry The use of mass spectrometry (MS) as a detection method in CE as well as EKC offers several advantages over the UV detection method. Analytes having no strong UV absorption are detected with a high sensitivity by MS detection. Furthermore, MS provides important information not only about the
MICELLAR ELECTROKINETIC CHROMATOGRAPHY
9
Detector S
BGE
(A) Analytes being swept Micelle vacancy S (B)
BGE
Anionic mcelle zone
BGE
BGE Completely swept analytes
(C)
Figure 6 Schematic diagram of the principle of sweeping under acidic conditions.
2.5 220 nm 1.5
mAU
0.5
DQ
− 0.5
s.p. PQ
DF
−1.5 − 2.5
EV
− 3.5 0
5
7
10 Time (min)
15
20 PQ
255 nm
6 5
mAU
4 3 2 DF
1
EV
0
DQ
s.p.
−1 −2 0
5
10 Time (min)
15
20
Figure 7 Electropherogram of tap water spiked with quaternary ammonium herbicides analyzed using CSEI-sweep-MEKC. Nonmicellar BGE, 100 mmol l 1 phosphate buffer (pH 2.5) containing 20% acetonitrile; micellar BGE, 80 mmol l 1 SDS in 50 mmol l 1 phosphate buffer (pH 2.5) containing 20% acetonitrile; high conductivity buffer (HCB), 200 mmol l 1 phosphate buffer (pH 2.5); conditioning solution before injection, nonmicellar BGE; sample concentration, 10 mg l 1 paraquat (PQ), diquat (DQ), and ethylviologen (EV, internal standard), 50 mg l 1 difenzoquat (DF). Injection scheme: hydrodynamic injection of HCB for 200 s (5 kPa), hydrodynamic injection of water for 6 s (5 kPa), electrokinetic injection of sample for 400 s ( þ 22 kV); separation voltage, 22 kV with the micellar BGE at both ends of the capillary. s.p., system peak. (Reprinted with permission from Nu´n˜ez O, Kim JB, Moyano E, Galceran MT, and Terabe S (2002) Journal of Chromatography A 961: 65–75. & Elsevier.)
molecular mass but also about the structure of the analytes. The most important issue in CE–MS may be the development of interfaces between CE and MS. MS has been successfully coupled to CE with various
interfaces. An electrospray ionization (ESI) interface is widely employed in CE–MS systems. However, the major drawback in MEKC–MS with an ESI interface is that the introduction of a nonvolatile PS into the
10
MICRO TOTAL ANALYTICAL SYSTEMS
interface reduces the ionization efficiency and contaminates the interface. To solve this problem, the use of the partial filling (PF) technique has been developed. In the PF technique, the micellar solution is filled only in a part of the capillary, and the analyte will interact with the micelle when passing through the micellar zone. In PF–MEKC, the applied voltage for the MEKC run will be cut off before the micellar zone reaches the end of the capillary or the interface to the mass spectrometer, so that the micelle is not introduced into the mass spectrometer. As an alternate ionization method to ESI, the atmospheric pressure chemical ionization (APCI) has been applied to the MEKC–MS system. In MEKC– APCI-MS, an SDS micellar solution can be introduced directly into the interface without a severe decrease in MS intensity. For highly sensitive analysis of environmental pollutants, an application study of sweeping to MEKC hyphenated with MS using an APCI interface has been reported. See also: Capillary Electrochromatography. Liquid Chromatography: Micellar. Solvents.
Further Reading Khaledi MG (1997) High-Performance Capillary Electrophoresis. New York: Wiley.
Kim JB and Terabe S (2003) On-line sample preconcentration techniques in micellar electrokinetic chromatography. Journal of Pharmaceutical and Biomedical Analysis 30: 1625–1643. Otsuka K and Terabe S (1989) Micellar electrokinetic chromatography. Bulletin of the Chemical Society of Japan 71: 2465–2481. Quirino JP, Kim JB, and Terabe S (2002) Sweeping: Concentration mechanism and applications toward high sensitivity analysis in capillary electrophoresis. Journal of Chromatography A 965: 357–373. Quirino JP and Terabe S (1999) Electrokinetic chromatography. Journal of Chromatography A 856: 465–482. Terabe S (1989) Electrokinetic chromatography: An interface between electrophoresis and chromatography. Trends in Analytical Chemistry 8: 129–134. Terabe S (1992) Micellar Electrokinetic Chromatography. Fullerton, CA: Beckman Instruments. Terabe S (1992) Selectivity manipulation in micellar electrokinetic chromatography. Journal of Pharmaceutical and Biomedical Analysis 10: 705–715. Terabe S, Chen N, and Otsuka K (1994) Micellar electrokinetic chromatography. Advances in Electrophoresis 7: 87–153. Terabe S, Otsuka S, and Ando T (1985) Electrokinetic chromatography with micellar solution and open-tubular capillary. Analytical Chemistry 57: 834–841. Terabe S, Otsuka K, and Ando T (1989) Band broadening in electrokinetic chromatography with micellar solutions and open-tubular capillaries. Analytical Chemistry 61: 251–260.
MICRO TOTAL ANALYTICAL SYSTEMS T McCreedy, University of Hull, Hull, UK & 2005, Elsevier Ltd. All Rights Reserved.
Introduction Miniaturized total analytical systems (mTAS) for chemical measurement have become important because they are reported to offer significant advantages over analytical systems of more conventional scale. These advantages are reported to include reduced reagent consumption and waste production, increased speed of analysis hence improved rates of sample throughput, and facilitating analysis in the field. There are other reported advantages with such systems, including the need for only very small volumes of sample (important where sample volume is limited) and disposability (particularly important for DNA replication work and clinical applications).
A common alternative name for mTAS is Lab-on-aChip. This article will describe typical mTAS, explain how such devices are made, and finally discuss a range of applications.
Total Analytical Systems and lTAS Chemical analysis does not consist solely of a physical measurement process providing a numerical value, e.g., concentration of pesticide, or identification, e.g., the unknown substance was 3,4-dinitrophenol. The actual measurement is one small part of a far more complex process commencing with sample collection and concluding with data interpretation. The complete analytical process can include filtration, pH adjustment, dilution, reagent addition, calibration, in addition to making the physical measurement and data processing. It has long been recognized that automating all these steps would
1
MICELLAR ELECTROKINETIC CHROMATOGRAPHY S Terabe and J-B Kim, University of Hyogo, Hyogo, Japan & 2005, Elsevier Ltd. All Rights Reserved.
Introduction Over the last two decades, capillary electrophoresis (CE) has been developed as a powerful separation technique for complex mixtures. Its advantages include a high separation efficiency, short analysis time, small sample requirement, and applicability to a wide range of analytes. The basic modes of CE that are presently being exploited include capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), capillary isotachophoresis, capillary isoelectric focusing, and capillary gel electrophoresis. In CZE, only ionic or charged analytes can be separated in principle since its separation mechanism is based on the difference in electrophoretic mobility of the analytes. MEKC has become popular as a powerful technique for the separation not only of neutral analytes but also of charged ones using a conventional CE instrument without any alteration. In MEKC, an ionic surfactant micelle is used as a pseudostationary phase (PS) that corresponds to the stationary phase in conventional chromatography and the surrounding aqueous phase to the mobile phase. The separation principle of MEKC is based on differential partitioning of analytes between the aqueous phase and the micelle phase. MEKC is a mode of electrokinetic chromatography (EKC) in which surfactants (micelles) are added to the buffer solution. They have polar head groups that can be cationic, anionic, neutral, or zwitterionic and nonpolar hydrocarbon tails. The formation of micelles is a direct consequence of the hydrophobic effect. The surfactant molecules can self-aggregate if the surfactant concentration exceeds a critical micelle concentration (CMC). The hydrocarbon tails will then be oriented toward the center of the aggregated molecules, whereas the polar head groups point outward. Micellar solutions can solubilize hydrophobic compounds that would otherwise be insoluble in water. Every surfactant has a characteristic CMC and aggregation number (AN), i.e., the number of surfactant molecules making up a micelle (typically in the range of 50–100) (see also Table 1). The size of the
micelles is in the range of 3–6 nm in diameter; therefore, micellar solutions exhibit properties of homogeneous solutions. Micelles have a dynamic structure that is the result of the rapid equilibrium between aggregated and monomeric forms. It is the differences in interaction between the micelle and the neutral solute that cause the separation. Micellar solutions have been employed in a variety of separations and spectroscopic techniques.
Micelles as Pseudostationary Phase for MEKC The CMCs and ANs of various kinds of surfactants suitable for PSs in MEKC are listed in Table 1. An anionic surfactant, sodium dodecyl sulfate (SDS), is most widely used as a PS in MEKC. Its popularity can be attributed to its low CMC, high aqueous solubility, low Krafft point, ready availability and the low cost of the pure product. Cationic surfactants such as tetradecyltrimethylammonium bromide (TTAB), cetyltrimethylammonium bromide (CTAB), and cetyltrimethylammonium chloride (CTAC) have also been useful for MEKC analysis. Most cationic surfactants have an alkyltrimethylammonium group, and their counterions are halides. The addition of cationic surfactants to the background electrolytes (BGE) caused the reversal of electroosmotic flow (EOF) owing to a positively charged capillary wall on account of the adsorption of cationic surfactants. As a result of the reversed EOF, the polarity of the electrodes has to be reversed in order to detect the analytes. Nonionic surfactants themselves do not possess electrophoretic mobility, but they have the distinct advantage of not contributing appreciably to Joule heating; hence, they may be used at high concentrations. They can have a great influence on the separation of charged analytes and can also be employed as PSs in mixed micelles with ionic surfactants. Nonionic surfactants such as polyoxyethylene (23) dodecyl ether (Brij 35), and polyoxyethylene (20) sorbitan monolaurate (Tween 20) have been effectively used as PSs for the separation of charged compounds through MEKC. Zwitterionic surfactants are not widely used in MEKC. However, zwitterionic surfactants will be interesting if they are used in mixed micelles or as a modifier of the micelle because they should show
2 MICELLAR ELECTROKINETIC CHROMATOGRAPHY Table 1 CMCs and ANs of selected surfactants
a
Surfactant
CMC a (10 3 mol l 1)
AN
Anionic SDS Sodium tetradecyl sulfate Sodium decanesulfonate Sodium N-lauroyl-N-methyltaurate Sodium polyoxyethylene dodecyl ether sulfate Sodium N-dodecanoyl-L-valinate Sodium cholate Sodium deoxycholate Sodium taurocholate Sodium taurodeoxycholate Potassium perfluoroheptanoate
8.1 2.1 (501C) 40 8.7 2.8 5.7 (401C) 13–15 4–6 10–15 2–6 28
62 138 40 – 66 – 2–4 4–10 5 – –
Cationic TTAB Dodecyltrimethylammonium bromide CTAB CTAC
3.5 15 0.92 1.3
75 56 61 –
Nonionic Polyoxyethylene(23) dodecyl ether (Brij 35) Polyoxyethylene(20) sorbitan monolaurate (Tween 20)
0.1 0.059
– –
Zwitterionic 3-((3-cholamiddopropyl)dimethylammonio)-1-propanesulfonate N-dodecyl-N,N-dimethylammonio-3-propanesulfonate
4.2–6.3 3.3
10 –
251C.
significantly different selectivities from other types of surfactants. Macromolecular surfactants or high-molecularmass surfactants (HMMS), butyl acrylate–butyl methacrylate–methacrylic acid copolymers (BMMAs), and sodium 10-undecylenate (SUA) oligomer have been introduced as PSs for MEKC. Since a HMMS forms a molecular micelle, which consists of one molecule, and the CMC value is essentially zero, one can expect a higher reproducibility in a HMMS– MEKC system compared with a low molecular mass surfactant (LMMS)–MEKC system. Bile salts are biological surfactants synthesized in the liver. The bile salts have both a hydrophilic and a hydrophobic face and tend to combine together at the hydrophobic face in an aqueous phase. To improve selectivity, various types of bile salts have been used as PSs in MEKC. Figure 1 shows the separation and determination of the ingredients of a cold medicine through MEKC with bile salt. Although they are not micelles, microemulsions and cyclodextrin (CD) derivatives have also been introduced as PSs for MEKC. Oil-in-water (o/w) microemulsions have been shown to be good PSs for EKC. Microemulsions (o/w) are prepared by mixing oil, water, a surfactant, and a cosurfactant such as a medium alkyl-chain alcohol. They have the characteristic properties of a solvent, such as thermodynamic stability and a high solubilization power. The
structure of the o/w microemulsion is similar to that of the micelle, except that the microemulsion has a core of a minute droplet of oil. The surfactant and the cosurfactant are located on the surface of the oil droplet to stabilize it. The separation basis of microemulsion EKC (MEEKC) is similar to that involved in MEKC. CD has been widely used not only in liquid chromatography but also in CE as a mobile phase modifier. The use of CD is particularly effective for the separation of aromatic isomers and enantiomers.
Fundamentals Separation Principle
Figure 2 illustrates a schematic representation of the separation principle of MEKC. When an anionic surfactant such as SDS is employed, the micelle migrates toward the anode by electrophoresis. The EOF transports the bulk solution toward the cathode due to the negative charge on the surface of fused silica. The EOF is usually stronger than the electrophoretic migration of the micelle under neutral or alkaline conditions, and therefore, the anionic micelle also travels toward the cathode at a retarded velocity. When a neutral analyte is injected into the micelle solution, a fraction of it is incorporated into the micelle and it migrates at the velocity of the micelle.
MICELLAR ELECTROKINETIC CHROMATOGRAPHY
3
6
8
2 7 10 + 11 14
9
3
5 1
4 12 13
0
5
10
15
20
25
Time (min) Figure 1 Separation of 14 ingredients through MEKC using bile salt. Buffer, 0.02 mol l 1 phosphate–borate (pH 9.0) containing 0.05 mol l 1 sodium deoxycholate; applied voltage, þ 20 V; temperature, ambient; detection wavelength, 210 nm. Solutes: 1, caffeine; 2, acetaminophen; 3, sulpyrin; 4, trimetoquinol hydrochloride; 5, guaifenesin; 6, naproxen; 7, ethenzamide; 8, phenacetin; 9, isopropylantipyrine; 10, noscapine; 11, chlorpheniramine maleate; 12, tipepidine hibenzate; 13, dibucaine hydrochloride; 14, triprolidine hydrochloride. (Reprinted with permission from Nishi H, Fukuyama T, and Terabe S (1990) Journal of Chromatography A 498: 313–323. & Elsevier.)
Detector
: Surfactant
: Electroosmotic flow (EOF)
: Solute
: Electrophoresis of micelles
Figure 2 Schematic illustration of the separation principle of MEKC.
4 MICELLAR ELECTROKINETIC CHROMATOGRAPHY Micelle
Analyte
Water
Capillary Injection (A)
Detector
Water
0
t0
Analyte
Micelle
tR
t mc
Migration time
(B)
Figure 3 Schematic diagram of the zone separation in MEKC (A) and chromatogram (B).
The remaining fraction of the analyte migrates at the velocity of the EOF, and thus the migration velocity of the analyte depends upon the distribution coefficient between the micellar and the nonmicellar (aqueous) phases. The greater the percentage of analyte that is distributed into the micelle, the slower it migrates. The analyte must migrate at a velocity between the electroosmotic velocity and the velocity of the micelle (see Figure 3), provided the analyte is electrically neutral. In other words, the migration time of the analyte, tR, is limited between the migration time of the bulk solution, t0, and that of the micelle, tmc. This is often referred to in the literature as the migration time window in MEKC.
eqns [4] and [5]: k¼
tR ¼ ð1 þ kÞ t0
k¼
nmc naq
½1
We can obtain the relation between the retention factor and migration time for an electrically neutral analyte as k¼
tR t0 t0 ð1 ðtR =tmc ÞÞ
½2
It can be rewritten as tR ¼
1þk t0 1 þ ðt0 =tmc Þ k
½3
When the migration time of the micelle is infinite or the micelle does not migrate in the capillary at all, t0/tmc will be zero; then eqns [2] and [3] become identical to those for conventional chromatography,
½4 ½5
In conventional chromatography, k ¼ N means that the solute is totally retained in the stationary phase and is not eluted at all or the retention time becomes infinite, whereas in MEKC k ¼ N means that the migration time of the solute, tR, is equal to tmc. In that case, such a solute migrates at the same velocity as the micelle or at the lowest velocity and is then detected last. When t0 ¼ 0 or the EOF is completely suppressed, eqn [3] becomes
Retention Factor
The retention factor, k, is defined as the ratio of the number of analytes incorporated into the micelle, nmc, and that in the surrounding aqueous solution, naq:
tR t0 t0
tR ¼
1 1þ tmc k
½6
In this case, the surrounding aqueous phase remains stationary in the capillary and the micelle migrates toward the anode by electrophoresis. Calculation of the retention factor using eqn [2] requires knowledge of t0, tR, and tmc. For the EOF marker of t0, methanol is often used since the distribution coefficient for methanol between the micelle and aqueous phases is negligibly small. Although methanol is UV transparent, it is detectable through the baseline disturbance of the UV detector due to the change in refractive index. Sudan III and Sudan IV are widely used as micelle markers since they are highly lipophilic and totally incorporated into micelle. Timepidium bromide, Yellow OB, and Orange OT are also useful as tracers of micelles. Note that eqn [3] was derived for the retention characteristics of neutral compounds. Hence, relationships between the migration time or mobility and retention factor will be more complicated when the
MICELLAR ELECTROKINETIC CHROMATOGRAPHY
solute has an electrophoretic mobility; that is, the migration time of the ionic solute includes a portion ascribed to the migration of the micelle when the solute is incorporated into the micelle and also another portion ascribed to the electrophoresis of the solute itself. Resolution
The resolution (Rs) in MEKC is given by eqn [7] Rs ¼
pffiffiffiffiffi N a1 k2 1 ðt0 =tmc Þ 4 1 þ k2 a 1 þ ðt0 =tmc Þ k1
½7
where N is the theoretical plate number, a is the separation factor equal to k2/k1, and k1 and k2 are the retention factors of two closely eluting analytes, 1 and 2, respectively. The resolution increases in proportion to the square root of the plate number. The higher the applied voltage, the higher the plate number, unless the conditions are such that the applied voltage generates excessive Joule heating. The average plate numbers for most analytes are usually in the range of 100 000–200 000. If the plate number is considerably lower, analytes are likely to be adsorbed on the capillary wall. In such cases, experimental conditions must be optimized to produce more efficient separations. Cleaning of the capillary is a possible procedure, as is changing the pH of the run buffer. Hydrophobic analytes, or those having longer migration times, typically yield high theoretical plate numbers because the micelle has a smaller diffusion coefficient. This is explained by assuming the major band broadening is due to the longitudinal diffusion as in CZE. The plate number does not depend significantly on the capillary length. With short capillaries, however, the amount of sample volume injected must be minimized to avoid zone broadening. The separation factor, a, is the most important and effective term for maximizing the resolution. The separation factor reveals the relative difference of the distribution coefficient between the two analytes and can be manipulated by chemical means. Since the distribution coefficient is a characteristic of a given separation system, the selectivity can be manipulated by changing either the type of micelle or by modifying the aqueous phase. The optimum value of the retention factor, kopt, for maximum Rs can be calculated by differentiating the following equation, provided N is independent of k: f ðkÞ ¼
k2 1 þ k2
1 ðt0 =tmc Þ 1 þ ðt0 =tmc Þ k1
½8
Then, kopt ¼
rffiffiffiffiffiffiffi tmc t0
5
½9
We can find that kopt is a function of tmc/t0. Under neutral conditions, the optimum value is B2 for SDS micelles as the PS. For practical use, the range of k recommended is between 1 and 5, or at maximum between 0.5 and 10. Unlike conventional chromatography, in MEKC the micelles as the PS migrate with the bulk liquid due to the EOF, in addition to the electrophoretic migration. This migration of the micelle through the EOF does not affect the separation at all. However, the distance of the migration of the micelle cannot be used for the separation. Since the time available for the solute to interact with the moving micelle depends on its k, the column availability depends on k as shown by the last term of the right-hand side of eqn [7] or [8].
Using Modifiers in MEKC MEKC is usually performed with a BGE that consists of just a buffer and a surfactant. Other substances may be added to the BGE to alter the selectivity. Modifiers may affect the charge on the micelle or the solute and change the distribution of the solute between the micelles and the surrounding aqueous phase by interacting with the solute. The following categories of additives are applicable in MEKC: CDs, ion-pair reagents, organic solvents, and others. Cyclodextrins
CDs are oligosaccharides with characteristic toroidal molecular shapes. Their outside surfaces are hydrophilic, while their cavities are hydrophobic. CDs tend to include molecules, which fit their cavities by hydrophobic interaction. The most widely used CDs consist of six (a-CD), seven (b-CD), or eight (g-CD) glucopyranose units. The size of the cavity differs significantly among the a-, b-, and g-CDs. Selectivity results from the inclusion of a portion of a hydrophobic solute into the cavity. The use of CDs is especially effective for the separation of aromatic isomers and aromatic enantiomers that have the chiral center close to the aromatic ring. Many CD derivatives have been developed for increased solubility in water as well as for modifying the cavity shape that provides different selectivities from underivatized CDs and are commercially available. In CD-modified MEKC (CD-MEKC), separation is achieved because of differences in the distribution of the solute among the micelle, CD, and the aqueous phase. The analyte molecule included by CD migrates at the same velocity as the EOF because
6 MICELLAR ELECTROKINETIC CHROMATOGRAPHY
CD electrophoretically behaves as the bulk aqueous phase. The neutral analyte molecule migrates at the same velocity as the EOF, whether included with CD or free from CD. On the other hand, the analyte migrates at a different velocity from that of the EOF when it is incorporated into the ionic micelle. Therefore, the addition of CD reduces the apparent distribution coefficient or k and enables the separation of highly hydrophobic analytes, which otherwise would be almost totally incorporated into the micelle. The higher the concentration of CD, the smaller the distribution coefficient. In CD-MEKC, therefore, the retention factor can be manipulated by varying either the concentrations of the micelle or CD. Enhancement of the separation selectivity of a group of polycyclic aromatic hydrocarbons (PAHs) using CD-MEKC using mixed CDs is shown in Figure 4. Ion-Pair Reagents
Ion-pair reagents are also very useful for selectivity enhancement of both ionic and nonionic analytes in MEKC. For example, when a tetraalkylammonium salt is added to an SDS micellar solution, the migration times of anionic analytes increase with an increase in the concentration of the ammonium salt
because the ammonium ion interacts with the anionic analyte to form a paired ion. Hence, the electrostatic repulsion between the anionic SDS micelle and the anionic analytes is reduced. On the other hand, a cationic analyte competes with the ammonium ion in interacting with the anionic SDS micelle, and so the migration times of cationic analytes decrease with an increase in the ammonium salt concentration. The effect of the ion-pair reagent on selectivity depends strongly on the structure of the reagent, e.g., the length of the alkyl chain. Organic Solvents
Organic solvents miscible with water are widely used as mobile phase modifiers in reversed phase high-performance liquid chromatography (RPLC). Organic solvents may also contribute to the enhancement of resolution or alteration of selectivity in MEKC. However, it should be noted that a high concentration of the organic solvent might break down the micellar structure. In general, the maximum content of the organic solvent is 20–30%. Methanol, 2-propanol, and acetonitrile are widely used as organic solvents in MEKC. The addition of organic solvents usually reduces the EOF and, hence, expands the migration time window. The retention factor is also reduced because the solubility of the analyte into the aqueous phase increases. Other Additives
13 12
1
10 7 9 11 14 2
5 6 3
8
18 1516 17
19 20
4 0
27 Time (min)
54
Figure 4 Separation of 20 PAHs using mixed CD-MEKC. Conditions: Capillary, 65 cm 50 mm inner diameter (50 cm length to the detector); injection by pressure, 0.02 min at 20 mbar, the concentration of each PAH in the mixture was 0.1 mg/ml; UV detection at 230 nm; separation solution, 0.042 mol l 1 b-CD, 0.026 g-CD, 2.5 mol l 1 urea, and 0.100 mol l 1 SDS in 0.140 mol l 1 borate buffer (pH 9); applied voltage, 15 kV. Solute identification: 1, acenaphthene; 2, acenaphthylene; 3, naphthalene; 4, benzo[g,h,i]perylene; 5, fluorene; 6, phenanthrene; 7, pyrene; 8, chrysene; 9, perylene; 10, anthracene; 11, benzo[e]pyrene; 12, benzo[a]pyrene; 13, benzo[a]anthracene; 14, fluoranthene; 15, dibenzo[a,h]pyrene; 16, benzo[k]fluoranthene; 17, triphenylene; 18, benzo[j]fluoranthene; 19, benzo[b]fluoranthene; 20, indeno[1,2,3-cd]pyren. (Reprinted with permission from Jime´nez B, Patterson DG, Grainger J, et al. (1997) Journal of Chromatography A 792: 411–418. & Elsevier.)
A high concentration of urea is known to increase the solubility of the hydrophobic compounds in water. Urea also breaks down hydrogen-bond formation in the aqueous phase. In MEKC, urea slightly reduces the electroosmotic velocity and considerably increases the electrophoretic velocity of the micelle, resulting in an expanded migration time window. The addition of urea is also effective in improving peak shapes, especially in the separation of amino acid derivatives. The selectivity is not remarkably changed by the addition of urea, but minor changes are noticeable, especially for the separation of closely related analytes. Another approach for changing selectivity in MEKC is the use of metal ions. In particular, the MEKC separation of oligonucleotides is improved by the addition of Mg(II), Cu(II), and Zn(II) ions. Metal ions are electrostatically attracted to the surface of a negatively charged micelle where they can be selectively complexed with analytes. Separation is due to differences in the distribution of solutes between the buffer and the metal–micelle surfaces. Retention is proportional to the complexation constant
MICELLAR ELECTROKINETIC CHROMATOGRAPHY
of the oligonucleotide and the metal–micelle surface. Using different metal ions can change the selectivity.
Improving Detection Sensitivity The most widely used detector in MEKC, which is common to other CE modes, is an ultraviolet (UV) photometric detector because many solutes have absorb UV, and the UV detector is easily set up and is cost efficient. One of the drawbacks of UV detection in MEKC is the low concentration sensitivity resulting from a short optical path length, equal to the capillary diameter, and the small injection volumes needed to maintain high efficiency. This limits the applicability of MEKC to trace analysis. Thus, method development is indispensable for reducing the limit of detection (LOD) or increasing the concentration sensitivity. There are different approaches that have been reported for improving the concentration sensitivity in MEKC. These investigations involve the use of highly sensitive detection methods, the installation of capillaries equipped with extended detection path lengths, and offline, and online sample preconcentration methods. The frequently used highly sensitive detection method in MEKC is laser-induced fluorescence (LIF). LODs for this detection mode are at least an order of magnitude lower than that for UV detection. However, in an ordinary laboratory these detectors may not be affordable and UV detection is widely used as previously mentioned. From Beer’s law, the absorbance is proportional to the path length, which is equal to the inner diameter of the capillary. Sensitivity can thus be enhanced through the use of larger inner diameter capillaries. However, this is not a good approach because large-diameter capillaries produce high currents, which cause Joule heating. The separation efficiency deteriorates dramatically when the capillary inner diameter is increased much above 100 mm. Because of problems with large inner diameter capillaries, most approaches to improving the sensitivity have been directed toward extending the path length without increasing the inner diameter of the entire capillary. Bubble cell, Z-shaped cell, and multireflection cell detector configurations have been introduced, which have afforded from twofold to more than 10-fold enhancements in detector response. As with HPLC, a successful MEKC analysis would often rely on dedicated offline sample preparation methods (e.g., liquid–liquid and/or solid-phase extraction). Sample preparation methods usually increase the concentration of the analyte before injection into the capillary. However, these methods require somewhat complex or expensive hardware, with limited applicability, or are time consuming.
7
On the other hand, online sample preconcentration methods based on electrokinetic focusing of large sample volumes represent one of the most facile ways of sensitivity enhancement in MEKC since the preconcentration step is performed within the same capillary used for separation. Two of the most widely used techniques are sample stacking and sweeping. In general, the application of online preconcentration techniques in MEKC is dictated by the specific properties of the analyte and the sample matrix conditions. Thereafter, a variety of online sample concentration techniques have been reported in CZE. They are field-enhanced sample stacking, largevolume sample stacking, transient-isotachophoresis (ITP), pH-mediated stacking, and dynamic pH junction. Sample Stacking
The sample stacking technique was first introduced for ionic analytes in CZE. The basic model of sample stacking is illustrated in Figure 5. In CZE (Figure 5A), the sample is dissolved in a solution that has a lower conductivity than that of the BGE and injected as a longer plug than in normal injection. When a separation voltage is applied, the electric field strength experienced in the sample zone is higher than in the rest of the capillary. The sample ions in the sample zone will move quickly and then slow down when they reach the BGE zone because they experience a lower electric field strength. Consequently, the ions are focused at the boundary of the two zones. It should be noted that the EOF is assumed to be zero in Figure 5A. However, this technique cannot be applied to neutral analytes because neutral analytes have no electrophoretic mobility. In MEKC (Figure 5B), to give effective electrophoretic mobilities to neutral analytes, charged PSs (e.g., anionic SDS micelles) are employed. The sample solution is prepared by dissolving the neutral analytes in a low-conductivity solution and injected into the capillary, which has been previously conditioned with an anionic SDS micellar BGE at neutral pH. The neutral analytes in the sample solution can be quickly carried to the boundary between the BGE and sample solution by the fast migrating anionic micelle entering the sample solution from the cathodic end. Since the electric field strength in the BGE zone is low, the velocity of the micelles is retarded, and the analytes are focused at the boundary between the BGE and the anodic end of the sample solution zone. Note that the neutral analytes are brought to the detector by the EOF since the magnitude of the EOF is greater than the electrophoretic velocity of the micelle.
8 MICELLAR ELECTROKINETIC CHROMATOGRAPHY Stacking boundary (SB) CZE Sample zone
BGS zone High− conductivity
Low−conductivity
Detector
BGS zone High− conductivity
(A) SB EOF MEKC Micellar BGS zone
Electrophoresis of micelles
N
N
Sample zone
N N
Micellar BGS zone
N N
(B)
E
Electric field strength
Figure 5 Schematic diagram of the principle of sample stacking in CZE (A) and MEKC (B).
However, a dispersive effect brought by the mismatch of local EOF velocities between the high and low electric field zones limits the injection length. To solve this problem, the stacking with reverse migrating micelles (SRMM) technique was developed. SRMM employs an acidic micellar BGE to reduce the EOF. Under this condition, the electrophoretic velocity of SDS micelles is higher than that of the EOF. Samples prepared in purified water or in low conductivity matrix are injected for a much longer time compared to the normal injection after conditioning the capillary with micellar BGE. Sample solutions are introduced at the cathodic end of the capillary, and then the separation voltage is applied with negative polarity at the injection end. Since the negative polarity is applied at the inlet, the sample matrix was slowly pushed out of the capillary by the weak EOF. Sweeping
The sweeping technique is defined as a phenomenon where analytes are picked up and concentrated by the micelle, which penetrates the sample zone devoid of micelle. It is independent of the EOF and effective for both charged and uncharged solutes. The principle of sweeping under the acidic condition is schematically shown in Figure 6. In step A, test analytes prepared in a matrix, with conductivity similar to that of BGE but devoid of PSs, are pressureinjected into the capillary at the cathodic end. In step
B, once the separation voltage is applied at the negative polarity with the BGE in the inlet vial, the anionic PS will enter the capillary and sweep the analytes. In step C, the analytes are completely swept by the PS and followed by MEKC separation in the reverse migration mode. Under the optimized condition, B5000-fold enhancements in detection sensitivity were obtained in terms of peak heights by sweeping. Cation- and anion-selective exhaustive injection sweeping (CSEI-sweep and ASEI-sweep) techniques are also available in MEKC. CSEI-sweep and ASEIsweep are combinations of two online preconcentration techniques, sample stacking with electrokinetic injection and sweeping, that can provide more than 100,000-fold enhancements in detection sensitivity. An electropherograms of tap water analysis by CSEIsweep-MEKC is illustrated in Figure 7. The results of this study suggest that CSEI-sweep-MEKC can be used for the analysis of quaternary ammonium herbicides in drinking water.
MEKC Hyphenation Technique with Mass Spectrometry The use of mass spectrometry (MS) as a detection method in CE as well as EKC offers several advantages over the UV detection method. Analytes having no strong UV absorption are detected with a high sensitivity by MS detection. Furthermore, MS provides important information not only about the
MICELLAR ELECTROKINETIC CHROMATOGRAPHY
9
Detector S
BGE
(A) Analytes being swept Micelle vacancy S (B)
BGE
Anionic mcelle zone
BGE
BGE Completely swept analytes
(C)
Figure 6 Schematic diagram of the principle of sweeping under acidic conditions.
2.5 220 nm 1.5
mAU
0.5
DQ
− 0.5
s.p. PQ
DF
−1.5 − 2.5
EV
− 3.5 0
5
7
10 Time (min)
15
20 PQ
255 nm
6 5
mAU
4 3 2 DF
1
EV
0
DQ
s.p.
−1 −2 0
5
10 Time (min)
15
20
Figure 7 Electropherogram of tap water spiked with quaternary ammonium herbicides analyzed using CSEI-sweep-MEKC. Nonmicellar BGE, 100 mmol l 1 phosphate buffer (pH 2.5) containing 20% acetonitrile; micellar BGE, 80 mmol l 1 SDS in 50 mmol l 1 phosphate buffer (pH 2.5) containing 20% acetonitrile; high conductivity buffer (HCB), 200 mmol l 1 phosphate buffer (pH 2.5); conditioning solution before injection, nonmicellar BGE; sample concentration, 10 mg l 1 paraquat (PQ), diquat (DQ), and ethylviologen (EV, internal standard), 50 mg l 1 difenzoquat (DF). Injection scheme: hydrodynamic injection of HCB for 200 s (5 kPa), hydrodynamic injection of water for 6 s (5 kPa), electrokinetic injection of sample for 400 s ( þ 22 kV); separation voltage, 22 kV with the micellar BGE at both ends of the capillary. s.p., system peak. (Reprinted with permission from Nu´n˜ez O, Kim JB, Moyano E, Galceran MT, and Terabe S (2002) Journal of Chromatography A 961: 65–75. & Elsevier.)
molecular mass but also about the structure of the analytes. The most important issue in CE–MS may be the development of interfaces between CE and MS. MS has been successfully coupled to CE with various
interfaces. An electrospray ionization (ESI) interface is widely employed in CE–MS systems. However, the major drawback in MEKC–MS with an ESI interface is that the introduction of a nonvolatile PS into the
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interface reduces the ionization efficiency and contaminates the interface. To solve this problem, the use of the partial filling (PF) technique has been developed. In the PF technique, the micellar solution is filled only in a part of the capillary, and the analyte will interact with the micelle when passing through the micellar zone. In PF–MEKC, the applied voltage for the MEKC run will be cut off before the micellar zone reaches the end of the capillary or the interface to the mass spectrometer, so that the micelle is not introduced into the mass spectrometer. As an alternate ionization method to ESI, the atmospheric pressure chemical ionization (APCI) has been applied to the MEKC–MS system. In MEKC– APCI-MS, an SDS micellar solution can be introduced directly into the interface without a severe decrease in MS intensity. For highly sensitive analysis of environmental pollutants, an application study of sweeping to MEKC hyphenated with MS using an APCI interface has been reported. See also: Capillary Electrochromatography. Liquid Chromatography: Micellar. Solvents.
Further Reading Khaledi MG (1997) High-Performance Capillary Electrophoresis. New York: Wiley.
Kim JB and Terabe S (2003) On-line sample preconcentration techniques in micellar electrokinetic chromatography. Journal of Pharmaceutical and Biomedical Analysis 30: 1625–1643. Otsuka K and Terabe S (1989) Micellar electrokinetic chromatography. Bulletin of the Chemical Society of Japan 71: 2465–2481. Quirino JP, Kim JB, and Terabe S (2002) Sweeping: Concentration mechanism and applications toward high sensitivity analysis in capillary electrophoresis. Journal of Chromatography A 965: 357–373. Quirino JP and Terabe S (1999) Electrokinetic chromatography. Journal of Chromatography A 856: 465–482. Terabe S (1989) Electrokinetic chromatography: An interface between electrophoresis and chromatography. Trends in Analytical Chemistry 8: 129–134. Terabe S (1992) Micellar Electrokinetic Chromatography. Fullerton, CA: Beckman Instruments. Terabe S (1992) Selectivity manipulation in micellar electrokinetic chromatography. Journal of Pharmaceutical and Biomedical Analysis 10: 705–715. Terabe S, Chen N, and Otsuka K (1994) Micellar electrokinetic chromatography. Advances in Electrophoresis 7: 87–153. Terabe S, Otsuka S, and Ando T (1985) Electrokinetic chromatography with micellar solution and open-tubular capillary. Analytical Chemistry 57: 834–841. Terabe S, Otsuka K, and Ando T (1989) Band broadening in electrokinetic chromatography with micellar solutions and open-tubular capillaries. Analytical Chemistry 61: 251–260.
MICRO TOTAL ANALYTICAL SYSTEMS T McCreedy, University of Hull, Hull, UK & 2005, Elsevier Ltd. All Rights Reserved.
Introduction Miniaturized total analytical systems (mTAS) for chemical measurement have become important because they are reported to offer significant advantages over analytical systems of more conventional scale. These advantages are reported to include reduced reagent consumption and waste production, increased speed of analysis hence improved rates of sample throughput, and facilitating analysis in the field. There are other reported advantages with such systems, including the need for only very small volumes of sample (important where sample volume is limited) and disposability (particularly important for DNA replication work and clinical applications).
A common alternative name for mTAS is Lab-on-aChip. This article will describe typical mTAS, explain how such devices are made, and finally discuss a range of applications.
Total Analytical Systems and lTAS Chemical analysis does not consist solely of a physical measurement process providing a numerical value, e.g., concentration of pesticide, or identification, e.g., the unknown substance was 3,4-dinitrophenol. The actual measurement is one small part of a far more complex process commencing with sample collection and concluding with data interpretation. The complete analytical process can include filtration, pH adjustment, dilution, reagent addition, calibration, in addition to making the physical measurement and data processing. It has long been recognized that automating all these steps would