CAPILLARY +L ELECTROPHORESIS
M e w 0. Ewing, Ross A. Wallllrgford, and Teresa M. Oleflrowlcr 152 Davey Laboratory Penn State University Universitv Park. PA 16802
The development of electrophoresis in capillary tubes offers several exciting methods for fast, highly efficient separations of ionic species, separations of macromolecules important in the area of analytical biotechnology, and development of small-volume separationsbased sensors. Initial work on electrophoresis in open tubes was presented in 1974, when Virtanen (I) reported the potentiometric detection of electrophoretically separated solutes in 200500-pm i.d. glass tubes. His work dealt with zone electrophoresis and discussed many of the unique advantages of using small-diameter tubes. Mikkers et el. (2) used this technique with 200pm i.d. Teflon tubes and obtained separations with plate heights less than 10 pm. The timely advance to smaller capillaries was made by Jorgenson and Lukacs ( 3 , 4 ) In . this work, plate heights of less than 1pm were predicted for separations of proteins and values of only a few micrometers were experimentally obtained for dansylated amino acids. Recently, Jorgenson (5)reviewed the area of analytically important electrophoresis; he emphasized traditional electrophoresis separation schemes for 282A
proteins and polynucleotides and demonstrated the potential of electrophoresis for analytical separations via preliminary results using zone electrophoresis in capillaries for the separation of proteins. This article will focus on the advances and impact of performing high-voltage electrophoresis in capillary tubes and will emphasize a description of our own research efforts in the area of separations-based sensors for analysis of microenvironments in biological systems. A more comprehensive review of capillary electrophoresis will be published elsewhere (6). The immediate potential for capillary electrophoresis methods lies in the area of analytical biotechnology, where there is a real need for both trace and micropreparative methods for protein and nucleic acid separations. Capillary zone, gel, and isoelectric focusing electrophoretic methods are all applicable in this area. There is also great potential for these methods in the area of separations-based sensors for ultrasmall-volume analysis. Here the separation method provides the selectivity step, and the detector provides the sensitivity. A key advantage in the use of capillary tubes for electrophoresis is an enhanced heat dissipation that permits the use of high potentials for separation. The use of high-potential fields leads to extremely efficient separations with a dramatic decrease in analysis time. In addition, several other advantages to the use of capillaries for elec-
ANALYTICAL CHEMISTRY, VOL. 61, NO, 4, FEBRUARY 15, 1989
trophoresis exist. The flow of solvent in a capillary when a tangential potential field is applied is termed electroosmosis (7). This flow, if not deliberately altered, is often strong enough to cause all solutes to elute at one end of the capillary. The presence of electroosmotic flow allows capillary electrophoresis to be more easily automated than its large-scale counterpart. Because a great deal of work has been done in the area of detectors for column liquid chromatography, technology is available to provide many very sensitive detection modes for electrophoresis in capillaries. Finally, the ultrasmallvolume flow rates obtainable in capillary electrophoresis permit sampling from picoliter environments. Indeed, the major thrust of our own work concerns the use of capillary electrophoresis to acquire and separate samples removed from the cytoplasm of single nerve cells.
Modes of capillaly electrophoresis The most frequently used modes of capillary electrophoresis have been capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, capillary gel electrophoresis, and isoelectric focusing. Isotachophoresis and isoelectric focusing were discussed in a prior REPORT (5) and will not be discussed in depth here. Isoelectric focusing of proteins in capillary tubes has been accomplished (8, 9). This technique involves placing a protein sample and a pH gradient 0003-2700/89/0361-292A1$01.50~0 0 1989 American Chemical SOCiety
0
0
0 0 forming solution into the capillary. Under the influence of an applied electric field, the charged proteins migrate to a region of pH where they are electrically neutral and will therefore stop migrating. After focusing, the zones are mobil i e d either by pumping or by electrophoresis of the zones through the capillary into a flow cell for detection. Resolution in isoelectric focusing is theoretically limited only by the slope and linearity of the pH gradient. Capillary zone electrophoresis. Zone electrophoresis in capillaries is analogous to elution chromatography techniques in that a narrow solute plug is introduced into a potential field. A schematic of a simple system used for capillary electrophoresis is shown in Figure la. A buffer-fdled capillary is placed between two buffer reservoirs, and a potential field is applied across this capillary. Ionic solutes then differentially migrate in a homogeneous buffer to provide discrete, moving zones. This is generally the simplest and perhaps the most universally useful mode of capillary electrophoresis. For most systems, electroosmotic flow is toward the cathode; hence, a detector is placed at this end. Injection of solutes is performed at the anodic end by either electromigration or hydrodynamic flow. Similarly to chromatographic techniques, capillary zone electiophoretic separations can be optimized with respect to efficiency, selectivity, and time. Electroosmosis is an important pro-
cess in capillary zone electrophoresis. Any solid-liquid interface is surrounded by solvent and solute molecules that are not oriented as in the bulk of solution. Figure l b shows a model of the silica-solution interface. Under normal aqueous conditions with small binary electrolytes, the solid surface has an excess of anionic charge resulting from ionization of surface functional groups. Counterions to these anions are in the stagnant double layer adjacent to the capillary walls. This cationic nature extends into the diffuse layer, which is
where E is the potential field strength. Because the flow originates at the diffuse region of the double layer, it is important to note the dimensions of this region. For a binary electrolyte in aqueous solution, the double-layer thickness ranges from 3 to 300 nm for electrolyte concentrations of to M, respectively. The extremely small size of the double layer leads to flow that originates at the walls of the capillary, resulting in a flat flow profile (Figure IC).Flat flow profiles in capillaries are expected when the capillary
mobile. The potential this creates across the layers is termed the zeta potential, and is given by Equation l:
radius is greater than seven times the double-layer thickness (12). In capillary zone electrophoresis, a flat flow profile and lack of need for a stationary phase result in a system of extremely high efficiency. Separations of dansylated amino acids that have been optimized for efficiency have been demonstrated with theoretical plate values that range from 2.7 X 106 for glycine to 3.3 X 106 for lysine (13). Losses in efficiency in capillary electrophoresis, however, can result when column heating, separation time, column geometry, injection and detection volumes, solute adsorption, and sample concentration are not optimized. Molecular diffusion is a large contributor to zone broadening in capillary
r,
where t~ is the viscosity, 6 is the dielectric constant of the solution, and F., is the coefficient for electroosmotic flow (10). The cationic counterions in the diffuse layer migrate toward the cathode, and, because these ions are solvated, they drag solvent with them. The extent of the potential drop across the double layer governs the rate of flow. The linear velocity, u, of the electroosmotic flow is given by Equation 2 (11):
ANALYTICAL CHEMISTRY, V o t . 61, NO. 4, FEBRUARY 15, 1989
293A
INSTRUMENTATION electrophoresis and can be minimized by designing separations where the solutes spend little time in the capillary (e.g., short columns, high voltages). Deviations can result in the flat flow profile if gravity flow is present; these can be minimized by assuring that each end of the capillary is at the same level so as not to produce a pressure differential across the column. Heating effects can be minimized by providing enough surface area to dissipate the heat generated, either through use of small inner-diameter capillaries, long capillaries, or a combination of the two. In general, when using 25-75-flm i.d. capillaries approximately 100 cm long, 2 5 3 0 kV represents an upper limit for the applied separation potential. Use of longer columns increases the surface area and hence heat dissipation; however, this also results in an increased analysis time. An alternative for minimizing thermal effects is active cooling of the capillary. The injection volume should be minimized to reduce overloading effects. Electric field distortion at high solute ion concentrations can also lead to elution of asymmetric zones. For best results, the concentration of buffer ions should be approximately lo00 times larger than that of the solute ions to minimize distortions in the applied electric field (4). Finally, interactions between solute ions and the capillary wall can result in significant tailing of peaks. In general, the existence of a negatively charged capillary wall leads to tailing of eluting cations. This can be minimized by adding salts to the operating buffer to compete for adsorption sites (14) or by deactivating the capillary surface by coating with an inert reagent (3, 15). Because many proteins strongly adsorb to the silica surface, strategies such 8% column wall deactivation, high sal buffers, and high pH buffers have heel used to optimize separations of proteins (3,9,14,15). Buffer selection is also important in capillary electrophoresis. The most common buffer system is 0.01-0.05 M phosphate at neutral pH. Much of the early work by Jorgenson and co-workers employed phosphate buffers in glass capillaries to produce excellent separations, mostly of anionic solutes. Buffer systems developed by Good (16) have become popular in capillary electrophoresis. These buffer systems are zwitterionic and therefore have a low conductivity. The pH and ionic strength of the buffer can also affect electroosmoticflow. Low ionic strength and high pH produce the fastest velocities in glass and fused-silica capillaries (17). 284A
many groups as a means of obtaining selective separations of neutral and ionic compounds while retaining the advantages of the capillary electrophoresis format. MECC is most commonly performed with anionic surfactants, especially sodium dodecyl sulfate (SDS). The MECC system is composed of two phases: aqueous and micellar. The surfaces of SDS micelles have a large net negative charge, giving them a large electrophoretic mobility toward the anode. However, most buffers exhibit a strong electroosmotic flow toward the cathode. The magnitude of eledroosmotic flow is slightly greater than that of micelle migration, resulting in a fastmoving aqueous phase and a slow-mov-
Capillary electrokinetic chromatography. In 1984 Terabe et al. (18) introduced the use of electroosmotically pumped micelles in a capillary electrophoresis system to affect chromatographic separations of neutral compounds. In this system, ionic surfactants are added to the operating buffer at concentrations exceeding the critical micelle concentration. At these levels, surfactant monomers tend to form roughly spherical aggregates, or micelles, with the hydrophobic tail groups oriented toward the center and the charged head groups along the outer surface. This technique, termed micellar electrokinetic capillary chromatography (MECC) by Burton et al. (19), has been extensively investigated by
Per,
Flgure 1. Schematics illustrating the capillary electrophoresis system and the basic principle of zone electrophoresis. (a) System for electrophoresis. (b) representation of surface and solvated ims at a silica-solution interface. and (0) representation of hflow profile resulllng from elecfrowmDtlc flow in capillaries.
ANALYTICAL CHEMISTRY, VOL. 61, NO. 4, FEBRUARY 15, 1989
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ing micellar phase. Solutes can partition between the two phases, resulting in retention based on differential solubilization by the micelles. Because the micellar phase is similar to a chromatographic stationary phase, the micelles have been termed a “pseudostationary phase”. The MECC system allows realization of the advantages of capillary LC without many of the drawbacks (i.e., stationary-phase coating technology) that have hindered the acceptance of that technique. The MECC system was originally developed for analysis of nonionic solutes, and retention in these systems is generally based on hydrophobicity. More hydrophobic solutes interact more strongly with the micellar phase and are therefore retained longer than hydrophillic compounds. Although MECC was developed as a means for separating neutral solutes, this technique can provide enhanced selectivity for separations of ionic species as well (20,21).The separation of neutral and ionic catechols shown in Figure 2 illustrates the selectivity for nonionic species, cations, and a zwitterion. Presumably, the nonionic species interact with the micellar phase based on their respective hydrophobicities, whereas the cationic and zwitterionic species interact with the charged Stern layer of the micelles. However, because the elution order of the cationic catecholamines is based on the relative hydrophobicities of the solutes, the surface interaction does not appear to be the only aspect controlling solubilization (21). The same cationic catechols are difficult to resolve by zone electrophoresis alone,
even in a system where 300,000 to 400,000 theoretical plates are generated (22).Thus the w e of micellar phases is clearly a useful means for controlling separation selectivity in the electrophoresis system. Capillary gel electrophoresis. An area of capillary electophoresis with great potential for protein separations is accomplished with the use of gelfilled capillary columns (15, 23). Gels are potentially useful for electrophoretic separations because they are an anticonvective media; they minimize solute diffusion, which contributes to zone broadening; they prevent solute adsorption to the capillary walls; and they eliminate electroosmosis,allowing maximum resolution in short lengths of column. Proteins can be separated based on their size using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) system (9,24).In the SDS-PAGE technique, proteins are denatured with 2-mercaptoethanol to promote unfolding. SDS added to the reduced proteins binds to the polypeptide chains through hydrophobic interactions. A constant amount of approximately 1.4 g of SDS is adsorbed by each gram of protein to form protein-SDS complexes with similar charge-to-size ratios. When electrophoresis of the protein-SDS complexes is performed in polyacrylamide gels (sieving gels), the complexes migrate with a velocity dependent only on the size of the complex (23). An example of a capillary SDSPAGE separation of myoglobin and several of its fragments is shown in Fig-
ure 3, along with a plot showing the dependence of mobility on molecular weight. The linear relationship between the logarithm of the molecular weight and the mobility allows determination of molecular weights of proteins falling within the range of standards. Efficiencies on the order of 40,000 theoretical plates allow for detection limits in the low-nanogram range when using UV detection. Analysis time can be manipulated by varying the monomer composition of the gel, changing the column length, or changing the operating voltage. Capillary SDS-PAGE holds several advantages over more conventional electrophoresis formats including nanogram sample capacity, prospects for automation, ease of quantitatiou, and sensitivity. Capillary gel electrophoresis with fraction collection has also been used for micropreparative purification of macromolecules ( 1 5 2 4 ) .It is anticipated that capillary gel electrophoresis will compliment slab gel techniques by providing researchers with a system capable of high throughput and two-dimensional separations (gel format), along with rapid and efficient molecular
fate-polyacrylamide gel electrophoresis separatlon of myoglobin and several fragments.
I
I
I
Condnlonl): 8S.S-m. 25pm 1.d. Iy8ed-silicacapillary: 5 mM Na&PoJ20 mM SOdlUm dcdacyl so1late, pH 7 butter: 20 kv applkd potential. Soiuies: A, Ldihydroxyphenyialaine: 8 , catechol: 6.Cmethylcatechol: D. norepinephine: E, eplnephrlne: F, 3.4-dlhydroxybeniylamine: G, dopamine. Solute A is a ZwMterion. solutes B and C are nonionic. and lutes D-0 are cations. (Adapted wilh permission from Reference 21.)
286A * ANALYTICAL
CHEMISTRY, VOL.
61. NO. 4. FEBRUARY 15. 1989
Conditions: 8 kV Separation: 2 k m , 75-pm 1.d. lusedsilica capillary: 0.1 M Tri%HsP04/0.1% S D W M urea, pH 6.9 buffer. Eluiion d e r : A. lragmem ill.MW 2510; B, lrag)mentII. MW 6210: C. fragment I, MW 8 1 8 0 D, lragmema 1 and ii, MW 14400; E. myoglobin. M W 17000. Inset: calibration plot 01 log MW vs. mobility for these species (Adapted wilh permission from Reference 23.)
~
weight determinations and trace qUantitation (capillary format).
R = (P,I - QJV/D(iie + d
mum separation efficiency (N)is given by
meory~oper~
where pa,l and k . 2 are the electrophoretic mobilities for the two solutes and & i s the average electrophoretic mobility (3, 4). Maximum resolution is obdried when p, = -iie; however, at this condition the analysis time should apDroach infmitv. Resolution is not independent of coiumn length at constant field strength (applied potential increasing with column length). Hence, longer columns with larger applied potential can be used to enhance resolution. This can also be carried out by balancing electroosmotic flow to be equal and opposite to ion migration, again resulting in a longer separation time. An important aspect of electroosmotic flow is that it permits automation of the system. However, because optimum resolution is obtained when electroosmotic flow is equal and opposite to electroDhoretic migration. an easily automaGd separationis not hkely to produce the best resolution.
This discussion will primarily concern aspects of free zone electrophoresis in capillary tubes. However, many of the points addressed are simila~for related capillary electrophoretic techniques. Separation efficiency. The total velocity of ionic solutes is in part dependent on electroosmotic flow. However, electroosmosis should not, in principle, affect the broadening of solute zones on the capillary for a given period of time. Because electroosmotic flow is flat. the main source of zone broadeningshould still be longitudinal diffusion (3.4). However, electroosmotic flow does affect the amount of time a solute resides in the capillary, and in this sense both the separation efficiency and resolution are related to the flow rate. In the presence of electroosmotic flow, the migration velocity Y and time t can be written as Y
= (Ire
+ r,)V/L
(3)
and where p. is the electrophoretic mobility, Vis the total applied voltage, and L is the length of the tube. A major limitation in normal or l a r g e - d e electrophoresis is solution heating owing to the ionic current carried between the electrodes. Joule heating can reault in density gradients and subsequent convection and temDerature madients that increase zone broadening, affect electrophoretic mobilities, and can even lead to evaporation of solvent. In large-scale electrophoresis, a supporting medium such as a gel is ueed to help dissipate heat, thereby minimizing these sour~esof band broadening. However, the support inereeses the surface area available for solute adsorption and introduces the band-broadening effect of eddy diffusion. A unique advantage of capillary tubs is the enhanced heat dissipation where heat is dissipated via the capillary wall. Maximized inner surface-area-to-volume ratios in smallbore capillaries provide more efficient heat dissipation relative to large-scale systams. The combined lack of any stationary phase and a tlat flow profde result in lonnitudinal 'diffusion as the major sourcs of band broadening in thii svstem. The abilitv to use hizh DOtentid fields (lWW-V/cm) p&des faster migration and flow rates, leading to rapid,-highly efficient separations-. Usinn Einstein's law of diffusion, the statk-tid equivalence of variance, and number of theoretical plates, the maxi298A
where D is the diffusion coefficient (3. 4). Component resolution. It can be noted from Equation 3 that if the rate of electroosmotic flow is greater in magnitude and opposite in direction to the electrophoretic migration velocity of all anions in the buffer, then all ions will move in the same direction. Additionally, nonionic species will be carried by the electroosmotic flow and elute at one end of the capillary. In thii system, electroosmotic flow carries the solutes and affects the total time merit in the capillary; however, separation is based on ditferential electroDhoretic migration. Therefore neutral- species are not readily separated by zone electrophoresis alone. In zone electrophoresis. the time that solutes spend in the capillary will be proportional to the real separation power of the system. The resolution R of two m e a in electrophoresis can be given by the equation
Deiecuon modso belng devekped
where N is the average number of theoretical plates, Au is the difference in zone velocities, and T is the average zone velocity (25).Using Eauations 3 and 5 and suhstituting into Equation 6, the resolution can be expressed aa
tble 1.
(7)
4Jz
Perhaps the most rapidly developing aspect of capillary electrophoresis is the area of detection. The ability to detect trace amounts of a wide variety of solutes will dictate the future of capillary electrophoresis. Although on-column UV absorbme and fluorescence have been the most commonly used detection modes. there has been a flurrv of new deteckon and derivatizatih schemes developed. Table I lists detec-
Detection modes developed for Capillary electrophoresis
Detection principle
Representative detecllon limite (moles detected)
Spectrophotometric
Representative reference(s)
,.
Absorptionb
."
"C
Fluorescence Precoiumn detivatization
On-column derivatization Postcolumn derivatization Indirect lluorescence Thermal lensb Ramanb
Mass spectrometric Electrochemical Conductivityb Potentiometric Amperometric
Radiometricb
x 10-19 i x 10-19
7
22,28
38
* Detection limit6 quoted have been determined with a wide variew of injection VOiUmes that range from 18 pL to 10 nL. a'&ass detection limit converfed Com concentration detection limit using a 1-nL injection volume.
ANALYfiCAL CHEMISTRY. VOL. 61,~NO.4, FEBRUARY 15, I989
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INSTRUMENTATION
tors with representative detection limits and references for the interested reader. The majority of these detectors will not be discussed here. Our group has emphasized the use of coupled capillaries for off-column detection by direct and indirect electrochemistry and mass spectrometry. These constitute only a minor portion of the work listed in Table I but will illustrate the principles being developed by many investigators in this area. Capillary electrophoresis-electrochemistry. We have developed a method to couple off-column detectors to capillary electrophoresis (22, 28). The simplest design uses electroosmotic flow to pump solutea past a break in the capillary at which the separation cathode is placed (Figure 4). In this system, the second segment of capillary has ground potential applied to the coupled end and hence displays very little interference from the high potential field on electrodes used for amperometric detection. The electrochemical
detector we have used consists of a 5- or 10-pm 0.d. carbon fiber electrode manipulated into the capillary and is similar to that designed for open-tubular liquid chromatography (39). Figure 5a shows an expanded electropherogram of several ionic catechols and a neutral catechol obtained on a 12.7-pm id. fused-silica capillary with amperometric detection. Examination of the peak widths at half height reveals that theoretical plate counts in excess of 800,000 can be achieved for small molecule separations in smallbore capillaries less than one meter long. Figure 5b shows an electropherogram of several ionic catechols and serotonin obtained on a 9-pm i.d. capillary (40). In this case, the high efficiency of the separation obtained permits the separation of serotonin and dopam i n e t w o cations having nearly identical electrophoretic mobilities. Also, peaks in this separation display a high degree of tailing because of adsorption
of the cationic solutes onto the anionically charged capillary walls. These effects are expected to be enhanced for smaller capillary diameters where the surface-area-to-volume ratio is maximized. However, the use of smaller cap. illary diameters leads to better mass detection limits and permits injection of even smaller volume samples. Results with 9-pm i.d. capillaries demonstrate detection limits (the signal-tonoise ratio is 2; peak-to-peak noise) of 7 X lo-'* mol of serotonin injected. In this example, the injection parameters corresponded to an 18-pL injection volume. Although the detection limit is volume-dependent, the concentration detection limit is 3.9 X 10-8 M for an 18-pL injection volume. Amperometric and laser-based detectors are among the most sensitive developed for capillary electrophoresis; however, most substances do not possea8 the properties necessary to use these detectors. Derivatization is one way to circumvent this problem. An al-
I
1 Micromanipulator Glass slide
I
!
Plexiglas block
riwn e. acnemaric or c a p i i i w eiecrropnoresis appararus wirn eiecrricaiiy conoucrive joinr ana amperomerric aerecrion sysrem. (a)Elscbophweaiasystem and (b) top view of detectim system. 300,.
ANALYTICAL CHEMISTRY, VOL. 61, NO. 4, FEBRUARY 15, 1989
(a) Conditions: 78.7-cm. 12.7-pm tusB6sIIica wparation capillary: 0.025 M MES, pH 5.55 buffer: 30 kV applied potential: 2 s Injection at 10 kV; 0.7 V vs. SSCE electrode potential: and 1.35 cm dnection capillary. Separation efficiencies are A, 838,000;8, 707.000; C. 549.000 D, 578.000 lheorelical plates based on peak hall-widlh for dopamine. epinephrine. isoproterenol. and hydroquinone, respectively. (b) CondRians: =me as (a) except 68.5-cm, 9-pm i.d. separation capillary and 1 s injscticm at 5 kV. Solutes: A. 150 m o l dopamine: 0.30am01 wrolonin; C. 160 a m i norepinephrine; D, 160 am01 epinephrine: E. 170 m o i isoprterenol: and F. 160 a m i Ldihyaoxyphenylalanine.(Adapted from Reference 40.)
ternative method is to use indirect detection schemes based on fluorescence or amperometry. The basic concept for indirect fluorescence detection involves use of an ionic fluorophore as a major constituent of the electrophoretic buffer. Ionic analvtes interact with the fluorophore, resilting in ion pairing or displacement of the fluorophore for oppositely charged versus similarly charged analyte ions, respectively. This results in a signal that is dependent on the properties of the fluorescent probe, and this signal is observed as a positive or negative peak for ion pairing versw displacement interactions, respectively. Kuhr and Yeung (31,32) demonstrated this concept by use of salicylate as the fluorescent probe for separation and detection of native amino acids by capillary electrophoresis coupled with laser fluorescence. We recently demonstrated capillary electrophoresis with indirect electrochemical detection. In these experiments, an easily oxidized substance, dihydroxybenzylamine, is used as a major constituent of the electrophoretic buffer. Ion pairing and displacement interactions results in analyte peaks for which detectability depends on the electrochemical properties of dihydroxybenzylamine and the concentration of ionic analyte. Figure 6 shows a capillary electro-
Figure I electrophoretic separation of amino acids with indirect electrochemical UW'LWCLIVII. Conditions: 101.5-m. 28.5-pm i.d. fused-siIica capillary: 0.9cm detection capillary: 1 X IO-' M dihydroxybenzyiamine/2.5 X M MES, pH 5.50 buffer; 20 kV applied potential; and 2 s injection ai 20 kV. Peaks: A, dihydroxybenzylaminedisplacement B. 138 Imoi lysine: C. 136 lmol arginine: D. 130 lmol histldine: E. 122 lmol lysinephenylalanine: F. 114 tmol histidine-phenyiaianins: S,System peak.
ANALYTICAL CHEMISTRY, VOL. 61. NO. 4, FEBRUARY 15. 1989
301 A
-
Electrochemical /detector
lure 7. System used for removal ani Planorbis corneus.
sin-
= J nerve cells of
(Adapted horn Reference 37.)
phoretic separation of three amino acids and two dipeptides detected by indirect electrochemistry. In this system, all the analytes and dihydroxybenzylamine are positively charged, resulting in negative peaks. The first peak to elute results from displaced electrophore, and the last peak is a system peak eluting at a time corresponding to electroosmoticflow. This system has not yet been optimized, and the detection limits observed range from 23 fmol for lysine to 49 fmol for lysinephenylalanine, leaving room for improvement. However, these initial results show great promise for expanding the sensitivity of amperometric detection to a broader range of solutes. Several other methods of detection currently are being developed. Laserbased fluorescence (with postcolumn derivatization), conductivity, and mass spectrometry are among the most promising. We are actively pursuing the concept of coupling fast atom bombardment mass spectrometry to capillary electrophoresis via our off-column detection procedure. With the advent of several of the above-mentioned detection schemes, capillary electrophoresis a p p e m to be well on the way toward fulfilling its potential.
Separatlons-based sensors One area of great promise in microcolumn separations is their use for analysis of discrete biological systems. Our research group has been involved in the development of capillary electrophoresis toward smaller capillary diameters for use as chemical sensors. The lowvolume capability, sensitive detection schemes, and use of electroosmotic flow for low-volume injection schemes make this a powerful approach to developing sensors for small biological environments. The main focus in this work is to permit determination of neurotransmitter concentrations in the cy802A
toplasm of single nerve cells. The principle of the sensor is selectivity by separation (capillary electrophoresis) and sensitivity by detection (electrochemi-
d). The neuronal aystem we have investigated is the giant dopamine cell of
Planorbis coreus (pond snail). This cell contains the easily oxidized neurotransmitter dopamine, which can be detected by electrochemistry. Dopamine stores in this cell are believed to be at least partially bound inside vesicles. Independent voltammetric studies using microvoltammetricelectrodes have shown that an easily oxidized sub0 m c e incream in concentration in the cytoplasm of this cell following exposure to an ethanol solution (41). The identification of this substance as dopamine is difficult by voltammetry, so a sample of cytoplasm was acquired via a microinjection system and solutes were separated by capillary electrophoresis (37,41).The system used for these experiments is shown in Figure 7. Injection of 100 to 300 pL of cytoplasm by electromigration into the microinjector was carried out at 55 s after exposure to ethanol. The resulting capillary electropherogram is compared with one for an authentic sample of dopamine in Figure 8.The migration times are nearly identical and provide
Flguro 8. Comparison of a standard electropherogram (top) to that 01 a sample taken from the giant dopamine neuron of Planorbis corneus after treatment with 100 pL 01 5 0 % ethanoll50% physiological solution (bottom). cOrddlw18:802rn. 14-Mrn .d. c~pillwy;eIBC11ochmlc~Idaeclion at 0.7V vs. SSCE; 0 025 M MES. pH 5.53 bdller: BO s cellul~rInJectlonSI 115 KV through a 9 . m o.d. mlcroinjeclu wim sampling inn olea 55 s aner emsnOl e x p o w ~ c aM 1 8 stsndard Wion a1 25 kV Imm 5 X M dcpammw.
ANALYTICAL CHEMISTRY, VOL. 61. NO. 4, FEBRUARY 15, 1989
further evidence that the substance increasing in concentration is dopamine. In addition, the peak area in this experiment corresponds to approximately 14 fmol of dopamine, which allows estimation of the cytoplasmic concentration at 4.6 X to 1.4 X lo-' M following ethanol exposure. This corresponds closely to the 9.7 X 10-5 M change in concentration observed by intracellular voltammetry (41). The use of capillary electrophoresis as the selectivity aspect of a chemical sensor has tremendous potential for the study of cellular and subcellular systems. The ultralow-volume capabilities, short analysis time, and high efficiencies available make this an ideal tool for this type of investigation. Use of this technique for cytoplasmic analysis is only in the initial demonstration stage, as is capillary electrophoresis in general. In the future, this concept will most certainly be expanded to peptide and protein separations from similar environments of pharmacological and physiological significance. In general, capillary electrophoresis is destined to be an important separation method where small-volume samples are involved and where quantitative electrophoresis of large biological molecules is necessary. Indeed, capillary electrophoresis should be useful in any case where fast, highly efficient liquidphase separations are required.
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Wegratefully acknowledge financial support from the National lnstitutes of Health, the National Science Foundation, Beekman Instruments, Sterling Pharmaceuticals. Monsanto Company. and Hoeehst-Roussell Pharmaceuticals. We would also like to acknowledge the many comments and suggestions made by Reginsldo Saraeeno in the writing of this manuscript.
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Ross A. Wallingford (left) is a senior chemist with Union Carbide Corp. in South Charleston, WV. After graduating from West Chester Uniuersity in 1984, he worked as a summer analytical research participant a t the Procter and Gamble Co. in Cincinnati, OH. He received his Ph.D. in analytical chemistry in 1988from Penn State Uniuersity under the direction of Andrew Ewing. His research interests include capillary electrophoresis, the application of new separation techniques to polymer analysis, pyrolysis gas chromatography, and the development of new detection schemes for separation techniques. Teresa M. Olefirowicz (center) is a second-year graduate student a t Penn State University and is pursuing a Ph.D. in analytical chemistry. She receiued her B.A. degree from Clark University in 1982. Prior to beginning graduate work, she worked for four years a t the Clorox Technical Center in Pleasanton, CA. Her research interests are in electrochemical and laser fluorescence detection for capillary zone electrophoresis and their application to intracellular neurochemical analysis. Andrew G. Ewing (right) is assistant professor of chemistry at Penn State Uniuersity and the recipient of a Presidential Younglnvestigator Award from the National Science Foundation. He receiued his B.S. degree from St. Lawrence University in 1979and his Ph.D. in analytical chemistry fromlndiana Uniuersity in 1983. He then spent 13 months as a postdoctoral fellow a t the Uniuersity of North Carolina-Chapel Hill. His research interests include electrochemistry at ultrasmall electrodes, capillary zone electrophoresis, and the application of these methods to neurochemical analysis. ANALYTICAL CHEMISTRY. V M . 61, NO. 4, FEBRUARY 15, 1989
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