Micellar Electrokinetic
Chromatography MEKC belongs to a mode of CE but also to micro-LC.
Shigeru Terabe University of Hyogo (Japan)
A
bout 20 years have passed since publication of the first paper on micellar electrokinetic chromatography (MEKC; 1), which is now widely accepted as a separation mode of CE (2). MEKC is particularly useful for separating small molecules, which traditionally has been impossible by gel electrophoresis. The separation mechanism is based on partitioning of the analyte between the micelle and the surrounding aqueous phase. This article demonstrates how MEKC separation can be reasonably controlled and how detection sensitivity can be improved.
+
–
Surfactant
EOF
Solute
Electrophoresis of micelles
FIGURE 1. Separation principle of MEKC.
© 2004 AMERICAN CHEMICAL SOCIETY
Fundamentals MEKC can be performed by dissolving an ionic surfactant in the CE running solution at a concentration higher than the critical micelle concentration (cmc) with no instrumental modifications. In general, neutral or alkaline buffer solutions are used to create conditions for a strong electroosmotic flow (EOF) that moves the entire liquid stream in the capillary toward the cathode (Figure 1). Therefore, even anionic micelles, such as sodium dodecyl sulfate (SDS), migrate toward the cathode. The neutral analyte that is not at all solubilized by or J U LY 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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is free from the micelle migrates at the same velocity as that of EOF; the analyte that is totally incorporated into the micelle migrates at the same velocity as that of the micelle. Other neutral analytes are detected between t0 and tmc, which are the migration time of the EOF marker and the micelle, respectively. The interval between t0 and tmc is called the migration time window. The wider the window, the larger the peak capacity, which is the number of peaks that can be separated during a run. Migration time can be measured by using markers such as methanol for EOF and Sudan III for the micelle. Parameters similar to those in chromatography can be used to describe the migration behavior of the analyte. The retention factor k can be defined as k = nmc/naq
tR – t0
(2)
t0 (1 – tR /tmc)
in which tR is the migration time of the analyte (1). The difference between this equation and the conventional one used in chromatography is the limited migration time window in MEKC. Although the micelle is not fixed inside the capillary, it plays the same role as the stationary phase in chromatography and is therefore called the pseudostationary phase.
Controlling selectivity and resolution In CE, the separation principle is simple and so is the strategy for optimizing separation conditions. Resolution is based on the difference in electrophoretic mobility and separation selectivity and is manipulated mainly by optimizing pH and, if necessary, by using additives to modify the electrophoretic mobility. Other issues are often more important, such as band broadening caused by the adsorption of analytes onto the capillary wall and low sensitivity due to a short optical pathlength in the photometric detector. These issues also occur in MEKC, but because MEKC separation is based on chromatographic separation, the optimization strategies are more versatile. The MEKC resolution RS equation is
k2 –1 RS = N · · 4 1+ k2
( )(
) · ( 1+1–(tt/t/t )·k ) 0
0
mc
mc
(3)
1
in which N is the plate number and is the selectivity factor equal to k2/k1 (3). Equation 3 is similar to the one used in conventional chromatography, except for the addition of the last term on the right-hand side. This variable comes from the migration of the micelle or pseudostationary phase inside the capillary; that is, the migration of the pseudostationary phase causes reduction of the column length (4). If the micelle migration is completely suppressed or tmc is infinity, the resolution equation is the same as the conventional one. 242 A
(b)
2 3
4
1
FIGURE 2. Micellar solubilization. (a) Ionic micelle and (b) mixed micelle of ionic and nonionic surfactants interacting (1) with the core, (2) on the surface, (3) as a cosurfactant, and (4) with a nonionic surface.
(1)
in which nmc and naq are the numbers of moles of the analyte in the micelle and surrounding aqueous phase, respectively; k can be measured by k=
(a)
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The plate number is not proportional to the capillary length as in other separation modes in CE under a constant applied voltage. Under conventional conditions, N is >100,000; if N is significantly lower than that, the experimental conditions must be reconsidered. The most probable cause of low N is adsorption of the analyte onto the capillary wall; if this happens, the capillary must be rinsed thoroughly. To manipulate the separation, must be changed (5). In reversed-phase LC, usually the separation is not manipulated by changing the stationary phase because C18 is the most widely accepted phase and different products vary little. In MEKC, however, pseudostationary phases are micelles, and several different surfactants can be used to form micelles. Some typical surfactants with their cmc and aggregation numbers are listed in Table 1. Using mixed micelles, especially combinations of ionic and nonionic or ionic and zwitterionic, creates other possible choices to change selectivity. The chemical structure of polar groups of surfactant molecules affects selectivity more than the hydrophobic core of the micelle or lipophilic groups of surfactant molecules, because most analytes interact with the micelle at the surface (Figure 2; 2). Mixed micelles (e.g., SDS and Brij 35) with surfaces covered by polyoxyethylene groups will have different surface characteristics and hence selectivity from that of the SDS micelle. According to the linear solvation energy relationship (6), hydrophobicity of the analyte is the major factor that determines selectivity. The second factor in tuning selectivity is the hydrogen-bond basicity of the analyte or hydrogen-bond acidity of the surfactant. Unlike the mobile phase in reversed-phase LC, the aqueous phase is not very important. In most cases, only the aqueous buffer is used, but if the analytes are extremely hydrophobic, up to 30% miscible organic solvents, such as methanol or acetonitrile, can be added to the micellar solution to increase solubility into the aqueous phase. Analyzing extremely hydrophobic analytes is a challenge in MEKC. The retention factor term (third term) in the right-hand side of Equation 3 is not independent of other variables because the last term includes k. The optimum k values to maximize resolution are easily determined by k opt =
tmc /t0
(4)
which differentiates the product of the last two terms in Equation 3 (7 ). Under neutral or acidic conditions, tmc /t0 is 3–4 and kopt is 1.7–2.0. To adjust k in MEKC, the concentration of the surfactant can be increased or decreased because k can be expressed as k = KVmc/Vaq K v– (Csf – cmc)
(5)
Bile salts, a group of readily available, natural surfactants that exhibit different selectivity as pseudostationary phases, can also be used. The micellar structure of bile salt is assumed to be very different from conventional long-alkyl chain surfactants. For example, many steroidal hormones are hydrophobic in nature but are easily separated by using sodium cholate. Using a high temperature may improve separation of extremely hydrophobic analytes because the distribution coefficient is generally low at high temperature.
in which K is the distribution coefficient of the analyte between the micelle and the aqueous phase; Vmc and Vaq are the volume of the micelle and the aqueous phase, respectively; v– is the par- Sensitivity enhancement tial specific volume of the micelle; and Csf is the surfactant con- Poor concentration sensitivity in CE is a serious problem and is centration (3). As shown by Equation 5, k is linearly propor- mainly due to the small amount of sample injected and a short tional to the surfactant concentration; this is an advantage of optical path length for absorbance detectors. One possible soMEKC, because the Csf needed to obtain a given k can be cal- lution is to use highly sensitive detectors for laser-induced fluoculated, provided the cmc and k at a certain Csf are known. In rescence or electrochemical measurements. However, these deTable 1, the cmc values are in pure water, but cmc in buffer so- tectors are expensive or may not be applicable because many analytes are not natively fluorescent or electrochemically active. lution is much lower. MEKC rarely separates extremely hydrophobic analytes with Another solution is to use extended optical path length cells high k. However, several strategies are possible. Adding an or- such as a bubble cell or Z-type cell, which can increase sensiganic solvent significantly reduces k and gives better resolution tivity 3–10-fold with a minimal decrease in resolution. A more promising choice for increasing concentration senof extremely hydrophobic analytes. The organic aqueous solution has higher viscosity, and the migration times will be long. sitivity is on-line sample preconcentration, in which a sample Adding too much organic solvent may destroy the micellar plug longer than normal is injected and focused inside the capstructure and/or decrease the migration time window because illary before separation. Either pressurized (also known as hydrothe electrophoretic mobility of the micelle is reduced, probably dynamic) or electrokinetic injection can be used. In the presas a result of the reduced charge on the micelle or the increased size of the micelle due to swelling caused by Table 1. Aggregation number (AN) and cmc of selected surfactants. the organic solvent. Surfactant AN cmc (at 25 °C; 10–3 M) Adding - or -cycloAnionic dextrin (CD) to the micellar SDS 62 8.1 solution is very effective. AlSodium tetradecyl sulfate 138 2.1 (50 °C) though CD itself is electriSodium decanesulfonate 40 40 cally neutral and behaves Sodium N-lauroyl-N-methyltaurate NA 8.7 like the aqueous phase, it Sodium polyoxyethylene dodecyl ether sulfate 66 2.8 can bind analytes in its caviSodium N-dodecanoyl-L-valinate NA 5.7 (40 °C) ty by hydrophobic interacSodium cholate 2–4 13–15 tion, depending on the size Sodium deoxycholate 4–10 of the analyte and the cavity. 4–6 In addition, CDs are chiral Sodium taurocholate 5 10–15 compounds and useful adSodium taurodeoxycholate NA 2–6 ditives for separating enanPotassium perfluoroheptanoate NA 28 tiomers, particularly neutral Cationic ones, in MEKC with achiral Tetradecyltrimethylammonium bromide 75 3.5 surfactants. Adding a high Dodecyltrimethylammonium bromide 56 15 concentration of urea inCetyltrimethylammonium bromide 61 0.92 creases the solubility of hyCetyltrimethylammonium chloride NA 1.3 drophobic compounds in Nonionic the aqueous solution and Polyoxyethylene(23) dodecyl ether (Brij 35) NA 0.1 decreases k. Urea is very Polyoxyethylene(23) sorbitan monolaurate (Tween 20) NA 0.059 soluble in water up to 7 M, Zwitterionic but the solution is UV3-[(3-cholamiddopropyl)dimethylammonio]-1-propanesulfonate 10 4.2–6.3 transparent and the viscosiN-dodecyl-N,N-dimethylammonio-3-propanesulfonate NA 3.3 ty is not very high. J U LY 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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electrophoresis. The sample plug length in pressurized injection and the sample injection time in FESI must be BGS BGS optimized for each analyte to maximize concentration Detector efficiency, because each analyte has a different electroInjection (a) Sample phoretic mobility related to k. [micelles] = 0 [micelles] > 0 In field-enhanced sample-stacking techniques, the difference in electrical conductivity (electric field strength) Micelles Micelles between the sample zone and the background solution (b) (BGS) zone, which contains the micelle, can be easily increased by preparing the sample solution in a low conductivity matrix. However, extreme differences in the [micelles] = 0 Concentration EOF of the two zones cause the focused sample zone to zone (c) mix at the boundary between the two zones. Therefore, acidic BGS is preferred for suppressing EOF in fieldenhanced sample stacking (11). The sample solution is usually prepared in a low electrical conductivity matrix, pure water, or aqueous organic solution, and the micelle is not added. However, this sample matrix is not comFIGURE 3. Sweeping with an anionic micelle under suppressed EOF conditions. patible with hydrophobic compounds, which tend to be (a) A sample solution prepared in a matrix with conductivity similar to that of BGS but efficiently concentrated because they are rarely soluble in devoid of micelle is injected hydrodynamically as a long plug. (b) Electrophoresis is started by applying voltage at negative polarity with the BGS in the inlet vial. The analytes are pure water. FESI solves this problem because the sample swept by the micelle penetrating the sample zone. (c) When the micelle from the inlet solution can be prepared with BGS that contains micelles vial reaches the boundary between the sample and the BGS zones, sweeping is finished (12). No matter which technique is used, maximum conand MEKC begins in the reversed migrating micelle mode. centration efficiency is ~100-fold, even under favorable conditions (13). Except for the fact that the sample mixture contains both surized method, the length of the maximum injection must be 4 pH units bepretreatment (19). CSEI and ASEI techniques are not applicable tween the sample and the BGS zones give better results. In printo neutral analytes. ciple, dynamic pH junction must be sensitive to the pKa of each Dynamic pH junction gives a several-hundred-fold increase in analyte; trying to concentrate many components in one run will J U LY 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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not produce good results. Additional studies need to be conducted to determine what kind of samples benefit most from dynamic pH junction. For samples that contain both ionic and neutral compounds, a combination of dynamic pH junction and sweeping provides superior on-line preconcentration (22). For a mixture of acidic and neutral analytes, sweeping with anionic micelle is appropriate; for a mixture of cationic and neutral analytes, a cationic micelle is appropriate. The sample zone must be devoid of micelle.
MS detection MS is a highly sensitive technique that provides useful information on molecular mass and structure. CE/MS is usually performed with an LC/MS system with or without a slight modification of the interface. Most CE/MS studies have used a sheath liquid-flow electrospray ionization (ESI) interface. These interfaces are convenient, easy to use, and do not require significant modification of the LC/MS system. The atmospheric pressure chemical ionization (APCI) interface is not popular in CE/MS because of the mismatch of the flow rate between LC and CE. In CE/MS, the total amount of the analyte injected is compatible with the amount of analyte required for MS analysis, but the sheath liquid dilutes the analyte >10-fold. Another problem in CE/MS is the buffer electrolytes. In a mass spectrometer, everything that enters the ionization chamber must be volatilized, but many electrolytes are nonvolatile, particularly the popular inorganic electrolytes such as phosphate or borate. Unfortunately, the number of MS-compatible electrolytes is limited; ammonium acetate and ammonium formate are popular. Several additives can manipulate selectivity, and most are nonvolatile, even in high vacuum. Using relatively high concentrations of surfactants in MEKC causes difficulties when it is interfaced with MS. In addition to contaminating the interface, several additives adversely affect ESI efficiency. To solve the contamination problem, the partial filling technique is used, in which only a portion of the capillary from the injection end is filled with BGS that contains the additive. Separation by interaction with the additive occurs only when the analytes pass the zone that contains the additive. The analytes keep migrating, without changing migration order, through the rest of the zone to the end of the capillary (23–25). The partial filling technique works very well with nonionic micelles, CDs, or proteins, but with ionic micelles such as SDS, the partial zone diffuses quickly and the migration time window narrows, making the technique unacceptable for MEKC/MS at this time. For now, the best procedure for MEKC/MS is to perform MEKC as usual with a slight loss of sensitivity (26). The surfactant concentration should be kept to a minimum, and a short analysis time should be used to avoid excessive contamination of the interface. If minor contamination of the interface is acceptable, APCI is a better choice because the loss of sensi246 A
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tivity caused by additives is negligible. However, no commercial APCI interface compatible with CE or MEKC is available yet. Further developments in instrumentation and improving MEKC/MS are strongly required. CE has not yet been widely accepted as a routine analytical separation technique, although it has several advantages over HPLC—less sample is required, and very little organic solvent is consumed. These advantages will become more prominent as time goes by. MEKC is now an established mode of CE, and several problems have been solved. Most analytical separations can be done by MEKC, except in the case of high-molecular-mass compounds such as proteins and oligosaccharides. In particular, on-line sample preconcentration enhances concentration sensitivity in MEKC more than it does in HPLC. CE/MS and MEKC/MS are not mature techniques, but more users will accelerate improvement of the instruments. Shigeru Terabe is a professor at the University of Hyogo (Japan). His research interests include separation sciences, particularly CE and microchip electrophoresis. Address correspondence about this article to him at the Graduate School of Material Science, University of Hyogo, Kamigori, Hyogo, 678-1297 Japan (
[email protected]).
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