Micellar Electrokinetic

Transcription

1 Micellar Electrokinetic

2 Chromatography MEKC belongs to a mode of CE but also to micro-lc. Shigeru Terabe University of Hyogo (Japan) About 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 Solute FIGURE 1. Separation principle of MEKC. EOF Electrophoresis of micelles 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 2004 AMERICAN CHEMICAL SOCIETY JULY 1, 2004 / ALYTICAL CHEMISTRY 241 A

3 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 t 0 and t mc, which are the migration time of the EOF marker and the micelle, respectively. The interval between t 0 and t mc 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 = n mc /n aq (1) in which n mc and n aq are the numbers of moles of the analyte in the micelle and surrounding aqueous phase, respectively; k can be measured by k = t R t 0 t 0 (1 t R /t mc ) in which t R 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 R S equation is 1 1 t 0 /t R = N k 2 mc S 4 ( ) ( 1+ k 2 ) ( 1+ (t 0 /t mc ) k 1 ) in which N is the plate number and is the selectivity factor equal to k 2 /k 1 (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 t mc is infinity, the resolution equation is the same as the conventional one. (2) (3) (a) 2 1 FIGURE 2. Micellar solubilization. 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 C 18 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 = t mc /t 0 3 (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. (b) 4 (4) 242 A ALYTICAL CHEMISTRY / JULY 1, 2004

4 which differentiates the product of the last two terms in Equation 3 (7). Under neutral or acidic conditions, t mc /t 0 is 3 4 and k opt is To adjust k in MEKC, the concentration of the surfactant can be increased or decreased because k can be expressed as k = KV mc /V aq Kv (C sf cmc) (5) in which K is the distribution coefficient of the analyte between the micelle and the aqueous phase; V mc and V aq are the volume of the micelle and the aqueous phase, respectively; v is the partial specific volume of the micelle; and C sf is the surfactant concentration (3). As shown by Equation 5, k is linearly proportional to the surfactant concentration; this is an advantage of MEKC, because the C sf needed to obtain a given k can be calculated, provided the cmc and k at a certain C sf are known. In Table 1, the cmc values are in pure water, but cmc in buffer solution is much lower. MEKC rarely separates extremely hydrophobic analytes with high k. However, several strategies are possible. Adding an organic solvent significantly reduces k and gives better resolution of extremely hydrophobic analytes. The organic aqueous solution has higher viscosity, and the migration times will be long. Adding too much organic solvent may destroy the micellar structure and/or decrease the migration time window because the electrophoretic mobility of the micelle is reduced, probably as a result of the reduced charge on the micelle or the increased size of the micelle due to swelling caused by the organic solvent. Adding - or -cyclodextrin (CD) to the micellar Anionic Surfactant solution is very effective. Although CD itself is electri- Sodium tetradecyl sulfate SDS cally neutral and behaves Sodium decanesulfonate like the aqueous phase, it Sodium N-lauroyl-N-methyltaurate can bind analytes in its cavity by hydrophobic interac- Sodium N-dodecanoyl-L-valinate Sodium polyoxyethylene dodecyl ether sulfate tion, depending on the size Sodium cholate of the analyte and the cavity. Sodium deoxycholate In addition, CDs are chiral Sodium taurocholate compounds and useful additives for separating enan- Potassium perfluoroheptanoate Sodium taurodeoxycholate tiomers, particularly neutral Cationic ones, in MEKC with achiral Tetradecyltrimethylammonium bromide surfactants. Adding a high Dodecyltrimethylammonium bromide concentration of urea increases the solubility of hy- Cetyltrimethylammonium chloride Cetyltrimethylammonium bromide drophobic compounds in Nonionic the aqueous solution and Polyoxyethylene(23) dodecyl ether (Brij 35) decreases k. Urea is very Polyoxyethylene(23) sorbitan monolaurate (Tween 20) soluble in water up to 7 M, Zwitterionic but the solution is UVtransparent and the viscosi- 3-[(3-cholamiddopropyl)dimethylammonio]-1-propanesulfonate N-dodecyl-N,N-dimethylammonio-3-propanesulfonate ty is not very high. 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. Sensitivity enhancement Poor concentration sensitivity in CE is a serious problem and is mainly due to the small amount of sample injected and a short optical path length for absorbance detectors. One possible solution is to use highly sensitive detectors for laser-induced fluorescence or electrochemical measurements. However, these detectors are expensive or may not be applicable because many analytes are not natively fluorescent or electrochemically active. Another solution is to use extended optical path length cells such as a bubble cell or Z-type cell, which can increase sensitivity 3 10-fold with a minimal decrease in resolution. A more promising choice for increasing concentration sensitivity is on-line sample preconcentration, in which a sample plug longer than normal is injected and focused inside the capillary before separation. Either pressurized (also known as hydrodynamic) or electrokinetic injection can be used. In the pres- Table 1. Aggregation number (AN) and cmc of selected surfactants. cmc (at 25 C; 10 3 M) (50 C) (40 C) AN JULY 1, 2004 / ALYTICAL CHEMISTRY 243 A

5 (a) Injection (b) (c) S BGS + BGS BGS Detector Sample [micelles] = 0 Micelles Concentration zone [micelles] = 0 [micelles] > 0 Micelles FIGURE 3. Sweeping with an anionic micelle under suppressed EOF conditions. (a) A sample solution prepared in a matrix with conductivity similar to that of BGS but 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 swept by the micelle penetrating the sample zone. (c) When the micelle from the inlet vial reaches the boundary between the sample and the BGS zones, sweeping is finished and MEKC begins in the reversed migrating micelle mode. surized method, the length of the maximum injection must be <90% of the effective capillary length (injector to detector) because at least 10% of the effective length must be left to separate the focused sample. In the electrokinetic method, more sample can be introduced because the injection zone is focused during the injection procedure. However, the size of the maximum injection must be found by trial and error. In any sample preconcentration technique, the focused zone length is usually narrower than that in conventional injection. If the size of the injection is not excessive and the real separation time is short, high resolution can be achieved, even in a short capillary. Several major on-line sample preconcentration methods have been reported in the literature, such as transient isotachophoresis (t- ITP; 8), electric field-enhanced (amplified) sample stacking (9, 10), sweeping, and dynamic ph junction. Among the four techniques, t-itp and dynamic ph junction are unsuitable for concentrating neutral compounds for MEKC. Although field-enhanced sample stacking was originally intended to preconcentrate ions, neutral analytes can be focused because they have an apparent electrophoretic mobility when they are incorporated into the micelle (11 13). The apparent electrophoretic mobility and the concentration efficiency are high for hydrophobic compounds. Two types of field-enhanced sample stacking techniques are available pressurized sample injection and field-enhanced electrokinetic sample injection (FESI), which are used for capillary zone + + electrophoresis. The sample plug length in pressurized injection and the sample injection time in FESI must be optimized for each analyte to maximize concentration efficiency, because each analyte has a different electrophoretic mobility related to k. In field-enhanced sample-stacking techniques, the difference in electrical conductivity (electric field strength) between the sample zone and the background solution (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 EOF of the two zones cause the focused sample zone to 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 compatible with hydrophobic compounds, which tend to be efficiently concentrated because they are rarely soluble in pure water. FESI solves this problem because the sample solution can be prepared with BGS that contains micelles (12). No matter which technique is used, maximum concentration efficiency is ~100-fold, even under favorable conditions (13). Except for the fact that the sample mixture contains both neutral and ionic analytes, sweeping is superior to stacking in MEKC (Figure 3). Sweeping is an original concentration technique created during the development of field-enhanced sample stacking of neutral analytes (14). Researchers found that having high electric field strength in the sample zone, which can cause adverse effects, is not required. They also found that the sample matrix can be prepared in a buffer solution with conductivity that is similar or higher than BGS and without adding micelle (15). Under these conditions, no enhanced field strength will be generated and electrophoretic migration and EOF will not change the velocity from the sample zone to the BGS zone. As also occurs in field-enhanced sample stacking in MEKC, analytes with higher k values are more efficiently concentrated in sweeping because of the distribution of the analyte into the micelle. In sweeping, EOF does not affect concentration efficiency because the field strength is homogeneous throughout the capillary and the mismatch of the EOF is low. However, maximum concentration efficiency was observed under suppressed EOF conditions. Sweeping is also very efficient and can provide up to a 5000-fold increase in detection sensitivity (14), endowing CE with concentration sensitivity that is higher than that of HPLC, even with a minute amount of sample. Sweeping has exhibited several advantages over sample stacking: Both neutral and charged analytes can be concentrated; high-concentration buffer can be used as a sample matrix; several different pseudostationary phas- 244 A ALYTICAL CHEMISTRY / JULY 1, 2004

6 (a) (b) es can be used as micelles; the presence of EOF does not adversely affect concentration efficiency; and a highconcentration electrolyte solution can be used as a sample matrix for analyzing biomedical samples. Strong interactions between the pseudostationary phase and the analyte can result in high concentration efficiency in sweeping but may cause poor resolution. Remember, however, that a strong interaction between the pseudostationary phase and the analyte is required only in the sample zone through which the micelle penetrates and that the BGS can contain additives to improve resolution. In most MEKC studies, SDS or anionic micelles can be used successfully to concentrate hydrophobic and cationic analytes. Cationic surfactants such as cetyltrimethylammonium bromide are used as a pseudostationary phase to concentrate anionic analytes such as carboxylates or sulfonates (16). Microemulsions and charged CDs are successful pseudostationary phases, and polyaminopolycarboxylates such as EDTA are used for sweeping metal ions through in-capillary complexation (17). FESI and sweeping were combined to give almost a millionfold increase in sensitivity (18). In the cation- or anion-selective exhaustive injection (CSEI or ASEI) sweeping method, cationic or anionic analytes are electrokinetically injected under field-enhanced conditions, and the long concentrated sample zone is focused by sweeping with the anionic or cationic micelle. The first step of electrokinetic injection consumes most of the analyte in the sample vial, and the sample concentration is sharply reduced. The sample solution cannot be used again. This technique is very useful for analyzing trace compounds in environmental samples. Figure 4 is an example of CSEI sweeping-mekc of herbicides in tap water at low parts-per-billion levels without any sample pretreatment (19). CSEI and ASEI techniques are not applicable to neutral analytes. Dynamic ph junction gives a several-hundred-fold increase in Time (min) FIGURE 4. Electropherogram of tap water analyzed by CSEI sweeping-mekc at (a) 220 nm and (b) 250 nm. 10 µg/l paraquat (PQ), diquat (DQ), and ethylviologen (EV, internal standard); 50 µg/l difenzoquat (DF); s.p., system peak. (Adapted with permission from Ref. 19.) s.p. s.p. concentration sensitivity and is based on the change in electrophoretic velocity between the sample zone and the BGS zone, which have different phs (20). For acidic analytes, the sample matrix is prepared at a ph that is lower than the pk a of the analyte and the BGS at a ph higher than the pk a. The acidic sample zone is gradually titrated by the alkaline BGS zone when the voltage is applied. The analyte is mostly neutral in the sample zone, and if it is in the neutralized boundary, it will be charged and will migrate by electrophoresis toward the injection end. The migration will stop when it reaches the acidic sample zone. It is difficult to describe precisely how the dynamic ph junction technique proceeds, although a computer simulation can provide helpful insight into the concentration mechanism (21). Finding the optimum concentration conditions is not easy. In addition to ph, the species of the buffer components and buffer concentration are important factors. Generally, significantly high concentration buffers and a difference of >4 ph units between the sample and the BGS zones give better results. In principle, dynamic ph junction must be sensitive to the pk a of each analyte; trying to concentrate many components in one run will DF DF EV EV DQ DQ PQ PQ 20 JULY 1, 2004 / ALYTICAL CHEMISTRY 245 A

7 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 sensitivity 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, Japan (terabe@sci.u-hyogo.ac.jp). References (1) Terabe, S.; et al. Anal. Chem. 1984, 56, (2) Otsuka, K.; Terabe, S. Bull. Chem. Soc. Jpn. 1998, 71, (3) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, (4) Zhang, C.-X.; Sun, Z.-P.; Lin, D.-K. J. Chromatogr. A 1993, 655, (5) Terabe, S. J. Pharm. Biomed. Anal. 1992, 10, (6) Yang, S.; Khaledi, M. G. Anal. Chem. 1995, 67, (7) Foley, J. P. Anal. Chem. 1990, 62, (8) Foret, F.; Szoko, E.; Karger, B. L. J. Chromatogr. 1992, 608, (9) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A 496A. (10) Beckers, J. L.; Bocek, P. Electrophoresis 2000, 21, (11) Quirino, J. P.; Terabe, S. Anal. Chem. 1998, 70, (12) Quirino, J. P.; Terabe, S. Anal. Chem. 1998, 70, (13) Quirino, J. P.; Terabe, S. J. Cap. Elec. 1997, 4, (14) Quirino, J. P; Terabe, S. Science 1998, 282, (15) Palmer, J.; Munro, N. J.; Landers, J. P. Anal. Chem. 1999, 71, (16) Kim, J.-B.; et al. J. Chromatogr. A 2001, 916, (17) Isoo, K.; Terabe, S. Anal. Chem. 2003, 75, (18) Quirino, J. P.; Terabe, S. Anal. Chem. 2000, 72, (19) Núñez, O.; et al. J. Chromatogr. A 2002, 961, (20) Britz-McKibbin, P.; Chen, D. D. Y. Anal. Chem. 2000, 72, (21) Kim, J.-B.; et al. Anal. Chem. 2003, 75, (22) Britz-McKibbin, P.; Otsuka, K.; Terabe, S. Anal. Chem. 2002, 74, (23) Nelson, E. M.; et al. J. Chromatogr. A 1996, 749, (24) Koezuka, K.; et al. J. Chromatogr. B 1997, 689, (25) Muijselaar, P. G.; Otsuka, K.; Terabe, S. J. Chromatogr. A 1998, 802, (26) Somsen, G. W.; Mol, R.; de Jong, G. J. J. Chromatogr. A 2003, 1000, A ALYTICAL CHEMISTRY / JULY 1, 2004