CAPILLARY ELECTROPHORESIS
Andrew G. Ewing, Ross A. Wallingford, and Teresa M. Olefirowicz 152 Davey Laboratory Penn State University University Park, PA 16802
The development of electrophoresis in capillary tubes offers several exciting methods for fast, highly efficient sepa rations of ionic species, separations of macromolecules important in the area of analytical biotechnology, and devel opment of small-volume separationsbased sensors. Initial work on electro phoresis in open tubes was presented in 1974, when Virtanen (1) reported the potentiometric detection of electrophoretically separated solutes in 200500-μπι i.d. glass tubes. His work dealt with zone electrophoresis and dis cussed many of the unique advantages of using small-diameter tubes. Mikkers et al. (2) used this technique with 200μτη i.d. Teflon tubes and obtained sep arations with plate heights less than 10 μτα. The timely advance to smaller cap illaries was made by Jorgenson and Lukacs (3,4). In this work, plate heights of less than 1 μπι were predicted for sepa rations 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 electro phoresis; he emphasized traditional electrophoresis separation schemes for
proteins and polynucleotides and dem onstrated the potential of electropho resis for analytical separations via pre liminary results using zone electropho resis in capillaries for the separation of proteins. This article will focus on the advances and impact of performing high-voltage electrophoresis in capil lary tubes and will emphasize a de scription of our own research efforts in the area of separations-based sensors for analysis of microenvironments in biological systems. A more comprehen sive review of capillary electrophoresis will be published elsewhere (6). The immediate potential for capil lary 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 poten tial for these methods in the area of separations-based sensors for ultrasmall-volume analysis. Here the sepa ration method provides the selectivity step, and the detector provides the sen sitivity. A key advantage in the use of capil lary tubes for electrophoresis is an en hanced heat dissipation that permits the use of high potentials for separa tion. T h e use of high-potential fields leads to extremely efficient separations with a dramatic decrease in analysis time. In addition, several other advan tages to the use of capillaries for elec
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trophoresis exist. T h e 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 c o u n t e r p a r t . Because a great deal of work has been done in the area of detectors for column liquid chromatography, technology is avail able to provide many very sensitive de tection modes for electrophoresis in capillaries. Finally, t h e ultrasmallvolume flow rates obtainable in capil lary electrophoresis permit sampling from picoliter environments. Indeed, the major thrust of our own work con cerns the use of capillary electrophore sis to acquire and separate samples re moved from the cytoplasm of single nerve cells. Modes of capillary electrophoresis The most frequently used modes of capillary electrophoresis have been capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, capillary gel electro phoresis, and isoelectric focusing. Isotachophoresis and isoelectric focusing were discussed in a prior R E P O R T (5) and will not be discussed in depth here. Isoelectric focusing of proteins in capil lary tubes has been accomplished (8, 9). This technique involves placing a protein sample and a p H gradient 0003-2700/89/0361-292A/$01.50/0 © 1989 American Chemical Society
forming solution into the capillary. Un der the influence of an applied electric field, the charged proteins migrate to a region of p H where they are electrically neutral and will therefore stop migrat ing. After focusing, the zones are mobi lized either by pumping or by electro phoresis of the zones through the capil lary into a flow cell for detection. Resolution in isoelectric focusing is theoretically limited only by the slope and linearity of the p H gradient. Capillary zone e l e c t r o p h o r e s i s . Zone electrophoresis in capillaries is analogous to elution chromatography techniques in t h a t 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-filled capillary is placed between two buffer reservoirs, and a potential field is applied across this capillary. Ionic solutes then differ entially migrate in a homogeneous buffer t o provide discrete, moving zones. This is generally the simplest and perhaps the most universally use ful mode of capillary electrophoresis. For most systems, electroosmotic flow is toward the cathode; hence, a detector is placed a t this end. Injection of sol utes is performed at the anodic end by either electromigration or hydrodynamic flow. Similarly to chromato graphic techniques, capillary zone electrophoretic separations can be opti mized with respect t o efficiency, selectivity, and time. Electroosmosis is an important pro
cess in capillary zone electrophoresis. Any solid-liquid interface is surround ed by solvent and solute molecules t h a t are not oriented as in the bulk of solu tion. 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 ex tends into the diffuse layer, which is
where Ε is the potential field strength. Because the flow originates at the dif fuse region of the double layer, it is important to note the dimensions of this region. For a binary electrolyte in aqueous solution, t h e double-layer thickness ranges from 3 to 300 nm for electrolyte concentrations of 10~ 2 t o Ι Ο - 6 Μ, 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 lc). Flat flow profiles in capil laries are expected when the capillary
INSTRUMENTATION mobile. T h e potential this creates across the layers is termed the zeta po tential, ξ, and is given by Equation 1: f = 4ττ^μ βο /ί
(1)
where -η is the viscosity, e is the dielec tric constant of the solution, and μβ0 is the coefficient for electroosmotic flow (10). The cationic counterions in the diffuse layer migrate toward the cath ode, 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 electroos motic flow is given by Equation 2 (11): u = (e/4irV)EÇ
(2)
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 t h a t have been optimized for efficiency have been demonstrated with theoretical plate values that range from 2.7 X 10 6 for glycine to 3.3 Χ 10 6 for lysine (13). Losses in efficiency in capillary electro phoresis, however, can result when col umn heating, separation time, column geometry, injection and detection vol umes, solute adsorption, and sample concentration are not optimized. Molecular diffusion is a large con tributor to zone broadening in capillary
ANALYTICAL CHEMISTRY, VOL 61, NO. 4, FEBRUARY 15, 1989 · 293 A
INSTRUMENTATION electrophoresis and can be minimized by designing separations where the sol utes spend little time in the capillary (e.g., short columns, high voltages). De viations can result in the flat flow pro file 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 dissi pate 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-μπι i.d. capillaries approximately 100 cm long, 25-30 kV represents an upper limit for the applied separation poten tial. Use of longer columns increases the surface area and hence heat dissi pation; 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 min imized to reduce overloading effects. Electric field distortion at high solute ion concentrations can also lead to elution of asymmetric zones. For best re sults, the concentration of buffer ions should be approximately 1000 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 add ing salts to the operating buffer to com pete for adsorption sites (14) or by de activating the capillary surface by coat ing with an inert reagent (3, 15). Because many proteins strongly adsorb to the silica surface, strategies such as column wall deactivation, high salt buffers, and high pH buffers have been used to optimize separations of pro teins (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-work ers 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 elec trophoresis. These buffer systems are zwitterionic and therefore have a low conductivity. The pH and ionic strength of the buffer can also affect electroosmotic flow. Low ionic strength and high pH produce the fastest veloci ties in glass and fused-silica capillaries (17).
Capillary electrokinetic chroma tography. In 1984 Terabe et al. (18) introduced the use of electroosmotically pumped micelles in a capillary elec trophoresis system to affect chromato graphic separations of neutral com pounds. In this system, ionic surfac tants 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 mi celles, with the hydrophobic tail groups oriented toward the center and the charged head groups along the outer surface. This technique, termed micellar electrokinetic capillary chromatog raphy (MECC) by Burton et al. (19), has been extensively investigated by
many groups as a means of obtaining selective separations of neutral and ionic compounds while retaining the advantages of the capillary electropho resis format. MECC is most commonly performed with anionic surfactants, especially so dium dodecyl sulfate (SDS). The MECC system is composed of two phases: aqueous and micellar. The sur faces 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 electroos motic flow is slightly greater than that of micelle migration, resulting in a fastmoving aqueous phase and a slow-mov-
Figure 1. Schematics illustrating the capillary electrophoresis system and the basic principle of zone electrophoresis. (a) System for electrophoresis, (b) representation of surface and solvated ions at a silica-solution inter face, and (c) representation of the flow profile resulting from electroosmotic flow in capillaries.
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INSTRUMENTATION ing micellar phase. Solutes can parti tion between the two phases, resulting in retention based on differential solu bilization by the micelles. Because the micellar phase is similar to a chromato graphic stationary phase, the micelles have been termed a "pseudostationary phase". The MECC system allows real ization of the advantages of capillary LC without many of the drawbacks (i.e., stationary-phase coating technol ogy) that have hindered the acceptance of that technique. The MECC system was originally de veloped for analysis of nonionic sol utes, 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 tech nique can provide enhanced selectivity for separations of ionic species as well (20,21). The separation of neutral and ionic catechols shown in Figure 2 illus trates the selectivity for nonionic spe cies, cations, and a zwitterion. Presum ably, the nonionic species interact with the micellar phase based on their re spective hydrophobicities, whereas the cationic and zwitterionic species inter act 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 generat ed (22). Thus the use of micellar phases is clearly a useful means for controlling separation selectivity in the electro phoresis 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 electrophore sis (SDS-PAGE) 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 polypep tide chains through hydrophobic inter actions. A constant amount of approxi mately 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 com plex (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 be tween the logarithm of the molecular weight and the mobility allows deter mination of molecular weights of pro teins falling within the range of stan dards. Efficiencies on the order of 40,000 theoretical plates allow for de tection limits in the low-nanogram range when using UV detection. Analy sis time can be manipulated by varying the monomer composition of the gel, changing the column length, or chang ing the operating voltage. Capillary SDS-PAGE holds several advantages over more conventional electrophoresis formats including nano gram sample capacity, prospects for automation, ease of quantitation, and sensitivity. Capillary gel electrophoresis with fraction collection has also been used for micropreparative purification of macromolecules (25, 24). It is antici pated that capillary gel electrophoresis will compliment slab gel techniques by providing researchers with a system ca pable of high throughput and two-di mensional separations (gel format), along with rapid and efficient molecular
Figure 3. Capillary sodium dodecyl sulfate-polyacrylamide gel electrophore sis separation of myoglobin and several fragments.
Figure 2. Electrokinetic separation of catechols. Conditions: 66.5-cm, 26-μιη i.d. fused-silica capillary; 5 mM Na2H2PO4/20 mM sodium dodecyl sulfate, pH 7 buffer; 20 kV applied potential. Solutes: A, L-dihydroxyphenylalanine; B, catechol; C, 4-methylcatechol; D, norepinephrine; E, epinephrine; F, 3,4-dihydroxybenzylamine; G, dopamine. Solute A is a zwitterion, solutes Β and C are nonionic, and solutes D-G are cations. (Adapted with permission from Reference 21.) 296 A · ANALYTICAL CHEMISTRY, VOL. 6 1 , NO. 4, FEBRUARY 15, 1989
Conditions: 8 kV separation; 20-cm, 75-μηη i.d. fused-silica capillary; 0.1 M Tris-H3PO4/0.1 % SDS/8 M urea, pH 6.9 buffer. Elution order: A, fragment III, MW 2510; B, fragment II, MW 6210; C, fragment I, MW 8160; D, fragments I and II, MW 14400; E, myoglobin, MW 17000. Inset: cali bration plot of log MW vs. mobility for these spe cies (Adapted with permission from Reference 23.)
INSTRUMENTATION weight determinations and trace quan titation (capillary format). Theory of operation
Ν=(μΒ + μ^)ν/2Ό
This discussion will primarily concern aspects of free zone electrophoresis in capillary tubes. However, many of the points addressed are similar for related capillary electrophoretic techniques. Separation efficiency. The total velocity of ionic solutes is in part de pendent on electroosmotic flow. How ever, electroosmosis should not, in principle, affect the broadening of sol ute zones on the capillary for a given period of time. Because electroosmotic flow is flat, the main source of zone broadening should still be longitudinal diffusion (3, 4). However, electroosmo tic flow does affect the amount of time a solute resides in the capillary, and in this sense both the separation efficien cy and resolution are related to the flow rate. In the presence of electroosmotic flow, the migration velocity ν and time t can be written as , = (Me + Meo)V/L (3) and 2
i = L /(Me + Meo)V
mum separation efficiency (iV) is given by
(4)
where μβ is the electrophoretic mobil ity, V is the total applied voltage, and L is the length of the tube. A major limitation in normal or large-scale electrophoresis is solution heating owing to the ionic current car ried between the electrodes. Joule heating can result in density gradients and subsequent convection and tem perature gradients that increase zone broadening, affect electrophoretic mo bilities, and can even lead to evapora tion of solvent. In large-scale electro phoresis, a supporting medium such as a gel is used to help dissipate heat, thereby minimizing these sources of band broadening. However, the sup port increases the surface area avail able for solute adsorption and intro duces the band-broadening effect of eddy diffusion. A unique advantage of capillary tubes is the enhanced heat dissipation where heat is dissipated via the capillary wall. Maximized inner surface-area-to-volume ratios in small bore capillaries provide more efficient heat dissipation relative to large-scale systems. The combined lack of any sta tionary phase and a flat flow profile result in longitudinal diffusion as the major source of band broadening in this system. The ability to use high po tential fields (100-900 V/cm) provides faster migration and flow rates, leading to rapid, highly efficient separations. Using Einstein's law of diffusion, the statistical equivalence of variance, and number of theoretical plates, the maxi
(5)
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. Addi tionally, nonionic species will be car ried by the electroosmotic flow and elute at one end of the capillary. In this system, electroosmotic flow carries the solutes and affects the total time spent in the capillary; however, separation is based on differential electrophoretic migration. Therefore neutral species are not readily separated by zone elec trophoresis 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 zones in electrophoresis can be given by the equation
*=f(f)
where Ν is the average number of theo retical plates, Av is the difference in zone velocities, and ν is the average zone velocity (25). Using Equations 3 and 5 and substituting into Equation 6, the resolution can be expressed as
Table I.
R m
foe,l ~ ^e,2)yWD(Me + μ J 4V2
where μθ,ι and μβ,2 are the electropho retic mobilities for the two solutes and ite is the average electrophoretic mobil ity (3, 4). Maximum resolution is ob tained when μ^ο = —jïe; however, at this condition the analysis time should approach infinity. Resolution is not independent of column 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 electrophoretic migration, an easily automated separation is not likely to produce the best resolution. Detection modes being developed
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 absorbance and fluorescence have been the most commonly used detection modes, there has been a flurry of new detection and derivatization schemes developed. Table I lists detec-
Detection modes developed for capillary electrophoresis
Detection principle Spectrophotometric Absorption" Fluorescence Precolumn derivatization On-column derivatization Postcolumn derivatization Indirect fluorescence Thermal lens" Raman6 Mass spectrometry Electrochemical Conductivity1' Potentiometric Amperometric Radiometric6
Representative detection limit9 (moles detected)
Representative reference(s)
icr 15 -icr 13
2, 18, 26
10- 1 7 -1(Γ 2 0 8 Χ 1(Γ 16 2 Χ 1(Γ 17 5 Χ 1(Γ 17 4 Χ 10-" 2 Χ 1(Γ 15 1 Χ 1(Γ 17
3,27 29 30 31,32 33 34 13,35
1 Χ 1(Γ 16 Not reported 7 Χ 1(Γ 19 1 Χ 10- 19
36 1 22,28 38
" Detection limits quoted have been determined with a wide variety of injection volumes that range from 18pLto 10 nL. 4 Mass detection limit converted from concentration detection limit using a 1-nL injection volume.
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INSTRUMENTATION tors with representative detection lim its and references for the interested reader. The majority of these detectors will not be discussed here. Our group has emphasized the use of coupled cap illaries for off-column detection by di rect and indirect electrochemistry and mass spectrometry. These constitute only a minor portion of the work listed in Table I but will illustrate the princi ples being developed by many investi gators 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 solutes 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 poten tial field on electrodes used for amperometric detection. The electrochemical
detector we have used consists of a 5- or 10-^m o.d. carbon fiber electrode ma nipulated into the capillary and is simi lar 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-|iim i.d. fused-silica capillary with amperometric detection. Examination of the peak widths at half height re veals that theoretical plate counts in excess of 800,000 can be achieved for small molecule separations in small bore capillaries less than one meter long. Figure 5b shows an electropherogram of several ionic catechols and se rotonin obtained on a 9-μπι i.d. capil lary (40). In this case, the high efficien cy of the separation obtained permits the separation of serotonin and dopa mine—two cations having nearly iden tical 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 ef fects are expected to be enhanced for smaller capillary diameters where the surface-area-to-volume ratio is maxi mized. However, the use of smaller cap illary diameters leads to better mass detection limits and permits injection of even smaller volume samples. Re sults with 9-μπι i.d. capillaries demon strate detection limits (the signal-tonoise ratio is 2; peak-to-peak noise) of 7 Χ 10 - 1 9 mol of serotonin injected. In this example, the injection parameters corresponded to an 18-pL injection vol ume. Although the detection limit is volume-dependent, the concentration detection limit is 3.9 Χ ΙΟ - 8 Μ for an 18-pL injection volume. Amperometric and laser-based de tectors are among the most sensitive developed for capillary electrophoresis; however, most substances do not pos sess the properties necessary to use these detectors. Derivatization is one way to circumvent this problem. An al-
Figure 4. Schematic of capillary electrophoresis apparatus with electrically conductive joint and amperometric detection system. (a) Electrophoresis system and (b) top view of detection system.
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Figure 5. Capillary electrophoretic separation with amperometric detection in small-bore capillaries. (a) Conditions: 78.7-cm, 12.7-μΓη fused-silica separation 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 detection capillary. Separation efficiencies are A, 838,000; B, 707,000; C, 549,000; D, 578,000 theoretical plates based on peak half-width for dopamine, epinephrine, isoproterenol, and hydroquinone, respectively, (b) Conditions: same as (a) except 68.5-cm, 9-μιτι i.d. separation capil lary and 1 s injection at 5 kV. Solutes: A, 150 amol dopamine; B, 30 amol serotonin; C, 160 amol norepinephrine; D, 160 amol epinephrine; E, 170 amol isopro terenol; and F, 160 amol L-dihydroxyphenylalanine. (Adapted from Reference 40.)
ternative method is to use indirect de tection schemes based on fluorescence or amperometry. The basic concept for indirect fluorescence detection in volves use of an ionic fluorophore as a major constituent of the electrophoret ic buffer. Ionic analytes interact with the fluorophore, resulting in ion pair ing or displacement of the fluorophore for oppositely charged versus similarly charged analyte ions, respectively. This results in a signal that is depen dent on the properties of the fluores cent probe, and this signal is observed as a positive or negative peak for ion pairing versus displacement interac tions, 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 electro phoresis coupled with laser fluores cence. We recently demonstrated cap illary electrophoresis with indirect electrochemical detection. In these ex periments, an easily oxidized sub stance, dihydroxybenzylamine, is used as a major constituent of the electro phoretic buffer. Ion pairing and dis placement interactions results in ana lyte peaks for which detectability de pends on the electrochemical proper ties of dihydroxybenzylamine and the concentration of ionic analyte. Figure 6 shows a capillary electro-
Figure 6. Capillary electrophoretic separation of amino acids with indirect electro chemical detection. Conditions: 101.5-cm, 26.5-/xm i.d. fused-silica capillary; 0.9-cm detection capillary; 1 Χ 10 - 4 M dihydroxybenzylamine/2.5 Χ 10" 2 M MES, pH 5.50 buffer; 20 kV applied potential; and 2 s injection at 20 kV. Peaks: A, dihydroxybenzylamine displacement; B, 138 fmol lysine; C, 136 fmol arginine; D, 130 fmol histidine; E, 122 fmol lysine-phenylalanine; F, 114 fmol histidine-phenylalanine; S, system peak. ANALYTICAL CHEMISTRY, VOL. 6 1 , NO. 4, FEBRUARY 15, 1989 · 301 A
INSTRUMENTATION
Figure 7. System used for removal and separation of cytoplasmic samples from sin gle nerve cells of Planorbis corneus. (Adapted from Reference 37.)
phoretic separation of three amino acids and two dipeptides detected by indirect electrochemistry. In this sys tem, all the analytes and dihydroxybenzylamine are positively charged, re sulting in negative peaks. The first peak to elute results from displaced electrophore, and the last peak is a sys tem peak eluting at a time correspond ing to electroosmotic flow. 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 im provement. However, these initial re sults show great promise for expanding the sensitivity of amperometric detec tion 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 bom bardment mass spectrometry to capil lary electrophoresis via our off-column detection procedure. With the advent of several of the above-mentioned de tection schemes, capillary electropho resis appears to be well on the way to ward fulfilling its potential.
toplasm of single nerve cells. The prin ciple of the sensor is selectivity by sep aration (capillary electrophoresis) and sensitivity by detection (electrochemi cal). The neuronal system we have inves tigated is the giant dopamine cell of
Planorbis corneus (pond snail). This cell contains the easily oxidized neuro transmitter dopamine, which can be detected by electrochemistry. Dopa mine stores in this cell are believed to be at least partially bound inside vesi cles. Independent voltammetric stud ies using microvoltammetric electrodes have shown that an easily oxidized sub stance increases in concentration in the cytoplasm of this cell following expo sure to an ethanol solution (41). The identification of this substance as do pamine is difficult by voltammetry, so a sample of cytoplasm was acquired via a microinjection system and solutes were separated by capillary electropho resis (37,41). The system used for these experiments is shown in Figure 7. Injection of 100 to 300 pL of cyto plasm by electromigration into the microinjector was carried out at 55 s after exposure to ethanol. The result ing capillary electropherogram is com pared with one for an authentic sample of dopamine in Figure 8. The migration times are nearly identical and provide
Separations-based sensors One area of great promise in microco lumn separations is their use for analy sis of discrete biological systems. Our research group has been involved in the development of capillary electrophore sis 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 de veloping sensors for small biological environments. The main focus in this work is to permit determination of neu rotransmitter concentrations in the cy
Figure 8. Comparison of a standard electropherogram (top) to that of a sample taken from the giant dopamine neuron of Planorbis corneus after treatment with 100 μ\. of 50% ethanol/50% physiological solution (bottom). Conditions: 80-cm, 14-μηη i.d. capillary; electrochemical detection at 0.7 V vs. SSCE; 0.025 M MES, pH 5.53 buffer; 60 s cellular injection at 115 kV through a 9-μιη o.d. microinjector with sampling initiated 55 s after ethanol exposure; and 1 s standard injection at 25 kV from 5 X 10~5 M dopamine.
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further evidence that the substance in creasing in concentration is dopamine. In addition, the peak area in this ex periment corresponds to approximate ly 14 fmol of dopamine, which allows estimation of the cytoplasmic concen tration at 4.6 X l f r 5 to 1.4 Χ 1(Γ 4 Μ following ethanol exposure. This corre sponds closely to the 9.7 Χ 10~5 Μ 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 capabil ities, short analysis time, and high effi ciencies available make this an ideal tool for this type of investigation. Use of this technique for cytoplasmic anal ysis 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 in volved and where quantitative electro phoresis of large biological molecules is necessary. Indeed, capillary electro phoresis should be useful in any case where fast, highly efficient liquidphase separations are required.
Chem. 1983,55,1365-70. (13) Smith, R. D.; Olivares, J. Α.; Nguyen, N. T.; Udseth, H. R. Anal. Chem. 1988,60, 436-41. (14) Lauer, H.; McManigill, D. Trends in Anal. Chem. 1986,5,11-15. (15) Hjerten, S.; Zhu, M.-D. J. Chromatogr. 1985,327,157-64. (16) Ferguson, W. J.; Good, N. E. Anal. Biochem. 1980,104, 300-10. (17) Lukacs, K. D.; Jorgenson, J. W. J. High Résolut. Chromatogr. Chromatogr. Commun. 1985,8,407-11. (18) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, Α.; Ando, T. Anal. Chem. 1984, 56,111-13. (19) Burton, D. E.; Sepaniak, M. J.; Maskarinec, M. P. J. Chromatogr. Sci. 1986, 24, 347-51. (20) Cohen, A. S.; Terabe, S.; Smith, J. Α.; Karger, B. L. Anal. Chem. 1987,59,102127. (21) Wallingford, R. Α.; Ewing, A. G. J. Chromatogr. 1988,441, 299-309. (22) Wallingford, R. Α.; Ewing, A. G. Anal. Chem. 1988,60, 258-63. (23) Cohen, A. S.; Karger, B. L. J. Chroma togr. 1987,397, 409-17. (24) Cohen, A. S.; Paulus, Α.; Karger, B. L. Chromatographia 1987,24,15-24. (25) Giddings, J. C. Sep. Sci. 1969, 4, 18189 (26) Walbroehl, Y.; Jorgenson, J. W. J. Chromatogr. 1984, 315,135-43. (27) Cheng, Y.-F.; Dovichi, N. J. Science 1988,242, 562-64. (28) Wallingford, R. Α.; Ewing, A. G. Anal. Chem. 1987,59,1762-66. (29) Pentoney, S. L., Jr.; Huang, X.; Burgi, D. S.; Zare, R. N. Anal. Chem. 1988, 60,
2625—29 (30) Rose, D. R., Jr.; Jorgenson, J. W. J. Chromatogr. 1988,447,117-31. (31) Kuhr, W.; Yeung, E. S. Anal. Chem. 1988,60,1832-34. (32) Kuhr, W. G.; Yeung, E. S. Anal. Chem. 1988.60, 2642-46. (33) Yu, M.; Dovichi, N. J. Anal. Chem. 1989.61, 37-40. (34) Chen, C.-Y.; Morris, M. D. Appl. Spectrosc. 1988,42, 515-18. (35) Olivares, J. Α.; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. Anal. Chem. 1987, 59, 1230-32 (36) Huang, X.; Pang, T.-K; Gordon, M. J.; Zare, R. N. Anal. Chem. 1987, 59, 274749. (37) Wallingford, R. Α.; Ewing, A. G. Anal. Chem. 1988,60,1972-75. (38) Gordon, M. J.; Huang, X.; Pentoney, S. L.; Zare, R. N. Science 1988,242, 22428 (39) Knecht, L. Α.; Guthrie, E. J.; Jorgen son, J. W. Anal. Chem. 1984,56, 479-82. (40) Wallingford, R. Α.; Ewing, A. G. Anal. Chem. 1989,61, 98-100. (41) Chien, J. B.; Wallingford, R. Α.; Ewing, A. G, submitted for publication in J. Neurochem. Suggested reading Hjerten, S. J. Chromatogr. 1983,270,1-6. Jorgenson, J. W. Trends in Anal. Chem. 1984,3, 51-4. Jorgenson, J. W. In New Directions in Electrophoretic Methods; Jorgenson, J. W.; Phillips, M., Eds. ACS Symposium Series 335; American Chemical Society: Wash ington, DC, 1987; pp. 182-98.
We gratefully acknowledge financial support from the National Institutes of Health, the National Science Foundation, Beckman Instruments, Ster ling Pharmaceuticals, Monsanto Company, and Hoechst-Roussell Pharmaceuticals. We would also like to acknowledge the many comments and suggestions made by Reginaldo Saraceno 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 University in 1984, he worked as a summer analytical research participant at the Procter and Gamble Co. in Cincinnati, OH. He received his Ph.D. in analytical chemistry in 1988 from Penn State University under the direction of Andrew Ewing. His research inter ests include capillary electrophoresis, the application of new separation tech niques 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 at Penn State University and is pursuing a Ph.D. in analytical chemistry. She received her B.A. degree from Clark University in 1982. Prior to beginning graduate work, she worked for four years at 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 neurochemi cal analysis. Andrew G. Ewing (right) is assistant professor of chemistry at Penn State University and the recipient of a Presidential Young Investigator Award from the National Science Foundation. He received his B.S. degree from St. Lawrence University in 1979 and his Ph.D. in analytical chemistry from Indiana University in 1983. He then spent 13 months as a postdoctoral fellow at the University 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, VOL. 61, NO. 4, FEBRUARY 15, 1989 · 303 A
