1884
AMI.
chem.1991, 63, 1884-1889
peak
peak excess = M 4 / M $ - 3
M4 = 3a4 + 6a2r2+ 9~~
The asymmetry ratio, bla, obtained from the peak width at 10% of the peak height, WO.lh, with a + b = WO.lh, was empirically related to the EMG 7 / u ratio by (19)
--4l
parameter change versus time. Observing Figures 2-5, it appears that the general shape of an exponentially modified perturbation (positive variation followed by a baseline return) is a tailing peak. This is the case when the underlying functions are as different as the square function (Figures 2 and 3), the triangle function (Figure 4), or the Gaussian function (Figure 5). It is possible that exponentially modified Gaussians are an ideal peak shape model. If it is not the case, the use of the EMG model to describe any tailing peak may be questionable. FIA peaks may be more accurately described and modeled by using exponentially modified square functions than EMG functions.
LITERATURE CITED PRACTICAL IMPLEMENTATION Programming the mathematical recursive series described by eq 8 is straightforward in any computer language. It needs few program lines. If the acquisition frequency is not constant, one more line shall be added to calculate the adequate parameter, A, by using eq ll with the desired constant value, 7 . Equation 8 is so simple that it is often not necessary to write a computer program to obtain the exponentially modified function. Modern electronic spreadsheets can be conveniently used. Figure 6 is a screen hardcopy of the spreadsheet used to draw Figure 4 with the software Lotus 123 (Lotus Development Corp., Cambridge, MA). Columns A and B contain the time, t, and signal, E @ ) ,respectively. Columns C-G contain the eq 8 convoluted signal, U t ) . The time constants, 7 , and corresponding A terms are contained in rows 1and 2, respectively. Cell D6 is highlighted to show (Figure 6, top left) how eq 8 is easily introduced in such a software. Changing a time constant in row 1 instantly produces the corresponding EMT values and graph. Tables I and I1 were obtained by using the same software.
(1) SaVbky, A.; W Y , M. J. E. Anal. Cbm. 1964. 36, 1627-1839. (2) Kkkland. J. J.; Yau. W. W.; Stoklosa, H. T.; Dilks, C. H. J. J . ChromeSCl. 1977, 15, 303-316. ( 3 ) Ruzlcka, J.; Hansen, E. H. Fbw In@tbn Anal)rslp: 2nd ed.:Chemlcal Analysis Serbs; Wlky: New York, 1988; Vol. 62. (4) Sternberg, J. C. I n Advances h chnwnetogaphy; Giddings, J. C., Keller, R. A., Eds.; Dekker: New York, 1960; Vol. 2, pp 205-270. (5) Gladmy, H. M.; Dowden. 8. F.; Swalen, J. D. Anal. Cl”.1969, 4 1 , 883-888. (6) Grushka, E. Anal. Chem. 1972, 44, 1733-1738. (7) Yau, W. W. Anal. Chem. 1077, 40, 395-398. (8) Pauls, R. E.: ROQWS,L. B. Anal. Chem. 1977. 40, 825-626. (9) Barber, W. E.; Can. P. W. AMI. Chem. 1981, 53, 1939-1942. (10) Fdey, J. P.; Dorwy, J. G. Anal. (XlCHn. 1963, 55, 730-737. J. 0. J . chnwnew. Scl. 1964, 22, 40-48. (11) FObY, J. P.: DO~SOY. (12) Fdey, J. P. AMI. Chem. 1967, 5 0 , 1984-1987. (13) Hemandez-Torres, M. A.; Khaledi, M. G.; Dorsey, J. 0. Anal. Chlm. ACIB 1967, 2Q1, 67-76. (14) B r W k S , S. H.; DOr~ey,J. 0. Anal. chkn. Acta 1990, 220, 35-46. (15) HanWl. D.; Can. P. W. AMI. Cbm. 1961, 57, 2394-2395. (16) Delley, R. chrometm@?k 1964. 18. 374-382. (17) M k y , R. Anal. chsm.1965. 57. 388. (18) Selby, M. S. StandardMeUmmatlCel Tabks; CRC Press: Boca Raton, FL, 1974. (19) Berthed, A. J . Liq. Chrometog. 1969, 12. 1187-1201. (20) Tyson, J. F. Anal. chkn.Acta 1966, 170, 131-148. (21) Grushka. E.; Myers, M. N.; Schetter, P. D.; Glddlngs, J. C. Anal. Chem. 1969, 41, 889-892. (22) Bldllngmeyer, B. A.; Warren, F. V. Anal. Chem. 1964, 56, 1583A1598A.
m.
CONCLUSION Equation 8 describes a simple generating function that can easily produce the EM form of any function representing a
RECEIV~,for review January 3,1991. Accepted May 30,1991.
CORRESPONDENCE Capillary Isotachophoresis with Concentration Gradient Detection Sir: Recently, capillary zone electrophoresis (CZE) has attracted significant attention from the analytical community (1,2). This method performs rapid, high-resolution separation of minute sample quantities and has important applications in biochemical and medical separations. Another interesting capillary electrophoretictechnique is capillary isotachophoresis (CITP) (3-8).CITP has certain advantages over CZE since it has less need for a sharp injection of high-concentration samples. Samples can be introduced with simple injection devices (3-5). Conventional detectors for CITP such as UV or conductivity offer sufficient sensitivity to monitor the high concentration of analytes in the separated zones. However, no high-spatial-resolution, sensitive detector has been proposed that would also allow the universal analysis of low0003-2700/91/0363-1884$02.50/0
concentration sample components. The major disadvantage of CITP is the necessity of using a discontinuous buffer system to separate anions or cations. A leading electrolyte (LE) and tailing electrolyte (TE) bracket the sample analytes and are chosen primarily on the basis of their mobility. The leading electrolyte has the highest mobility and migrates first in the separation capillary and is followed by the sample zones and the tailing electrolyte. During the course of the separation, each sample component becomes separated into a pure zone, stacked sequentially between the leading and tailing electrolytes, according to its mobility and degree of ionization at a given pH. After the steady state is reached, the zones migrate toward the detector. For example, if a sample originaUy contains two components S1and S2,then, 0 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63,NO. 17, SEPTEMBER 1, lQQl
6
A
1865
-dn =-- dndC dx dC dx
It is the refractive index gradient with associated Concentration gradient that is detected by the concentration gradient detector. In this universal method of detection, a laser beam is focused into the capillary. The large change of the refractive index between two zones produces a deflection of the beam 9 toward higher refractive index, which generates the signal
B T
e = L- d- n ndx
X
I
-
X
El
X
Flgura 1. Capbry isatachophoretlc Separation of two catkns. Leading ion is ammonium ion (10 mM), tailing is tetraoctylammonium ion (4.5 mM), sample components S, hexadecyitrimethylammnium ion (3.0 mM) and S, ethykriphenyiphosphonium ion (2.8 mM). (A) Two components are separated into pure zones between LE and TE producing narrow boundaries. (B) Hypothetical trace corresponding to refractive index detectkn. (C) Refractive index gradient detection of two zones. Applied voltage is 3.5 kV. (D) Integral of the trace from C.
in the steady state of the separation, two pure zones, one containing SIand the other containing S2,will form as shown in Figure 1A. The leading and tailing electrolytes will also form distinct zones, creating in total four zones with three sharp boundaries between them. The separated zones of each pure sample component have associated characteristic propertiea such as refractive index, temperature, conductivity, and electric field strength. Therefore, corresponding universal sensors can be designed to measure the changes in these parameters and to monitor the arrival of each zone. For example, a refractive index detector could be applied to the separation shown in Figure lA, to yield the hypothetical trace given in Figure 1B. The isotachopherogram will consist of “steps” indicating differences in refractive index between zones. This could be practically implemented by using the refractive index detector based on light-diffraction principle in ita traditional way (9) or a more modem configuration (10). After the steady state is reached in the CITP separation, narrow boundaries are formed between zones. They remain narrow during the entire separation because of the selfsharpening effect, which eliminates the effects of diffusion (3-6). High-concentration gradients (dC/dz) are produced at the boundaries between zones. These concentration gradients generate a corresponding refractive index gradient (dn/ dx):
where L is the diameter of the separation capillary and n is refractive index (11,121. For example, Figure 1C illustrates the trace that is produced when the concentration gradient detector is applied to detect the three boundaries formed in a CITP separation of a mixture of two cations between a leading (LE) and a tailing (TE) electrolyte. As expected, the first derivative of the refractive index profile from Figure 1B is obtained. The gradient trace can be either positive or negative, depending on the direction of change of the refractive index across the boundary. The three zone boundaries are detected with high sensitivity by the gradient detector. Only changes in the electrolyte composition at the boundaries are seen, not absolute zone properties. The intense signals produced with the gradient method are in direct contrast to the small signals that would be produced by refractive index detection as shown on Figure 1D. Figure 1D is generated by integrating the refractive index gradient trace from Figure 1C. Contrary to the hypothetical trace of Figure lB, the actual refractive index detection is subject to low frequency noise, which is most likely associated with temperature fluctuations. These drifts can seriously hinder the detection especially when the difference in refractive index between two adjacent zones is small, for example, the third boundary in Figure 1D. The derivative nature of the gradient detector removes the low frequency noise associated with theae drifts and produces a “flat baseline”, as shown in Figure 1C (13,14). In this paper, we present the results and observations obtained during our initial investigations of capillary isotachophoresis with the concentration gradient detector.
EXPERIMENTAL SECTION Figure 2 shows the isotachophoresis system, consisting of an injector and a separation capillary, and the concentration gradient detection system, consisting of a laser, a focusing lens, and a position sensor. The laser beam is focused directly into the separation capillary and then intercepted by the position sensor. Isotachophoresis Apparatus. A sliding Teflon injector was designed for sample injection in CITP and is described in detail in ref 15. The injector is based on a sliding mechanism and has two positions, fill and inject. In the fill position, the sample, LE, and TE are rinsed through the injector. The sample fills a hole drilled into the barrel of the injector. In the inject position, a 7-pL sample, contained in the barrel, is introduced between the LE and TE. The separation capillary is made of a copolymer of tetrafluoroethylene and hexafluoropropylene(FEP, Cole Parmer, Chicago, IL) tubing, 0.88-mm inner diameter. The total length of the capillary is 35.0 cm, and the length from the injector to the detector is about 11.0cm. Membranes (Type EPA, Osmonica, Minnetonka, MN) are used to prevent hydrodynamic flow and are attached to reservoir containers at either end of the capillary. The capillary is mounted on a three-stage optical mount (Model MR3 linear XYZ translation stage, Klinger Scientific, Montreal, Quebec, Canada) and placed on a vibration isolation table. The current is monitored at the grounded end of the separation capillary, by measuring a drop of voltage across a 1-kflresistor, using a digital multimeter (HM 8011-2,Hameg, West Germany). A voltage-stabilized, high-voltage power supply (Spellman,
1886
ANALYTICAL CEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
ANODE IN CAPlM MEMBRANE LEADING ELECTROLYTE
I
INJECTOR
POSITION SENSOR
TAILING ELECTROLYTE
MEMBRANE-
b
CATHODE
Figure 2. Experimental arrangement.
Plainview, NY)is uaed to drive the separation. This type of the power supply allowed determination of the boundaries positions with accuracy better then 25% by calculating the average velocity of the ions in the capillary as discussed below. A more accurate determination of the zone dimension requires continuous monitoring of the current or use of a current-controlledpower supply, which facilitates the constant velocity of the isotachophoretic assembly throughout the separation. Parts of the instrument that are exposed to the high voltage, are shielded by a Plexiglas box. The potential is applied to platinum wires embedded into the buffer reservoirs. The negative end of the capillary is grounded. Gradient Detection. The optical arrangement consists of a light source that is a helium-neon laser (Model 1303p, Uniphase, San Jose, CA) or a laser diode (MWK Industries, Pomona, CA) and photodiode position sensor. The laser beam is focused to a small spot size ( d ) of 40-50 pm directly into the separation capillary. This focal spot diameter is calculated from the following equation: d = -4AF
m
(3)
where X is the wavelength of the probe beam (632.8nm for HeNe laser beam), F is the focal length of the lens used to focus the beam (in our case F = 38.10 mm), and D is the diameter of the laser beam. The probe beam is arranged so that the far field intensity profile points to the center between two photodiodes placed close together (16). When irradiated uniformly, the photodiodes generate equal amounts of photocurrent. Upon encountering a concentration gradient, the laser probe beam is deflected and the amount of light reaching the diodes is not equal. The difference in photocurrent associated with the two diodes corresponds to the magnitude of deflection of the laser beam. The difference in photocurrent generated by the diodes is converted to a voltage by a single operational amplifier (16). The data are collected by an IBM DACA board, in a PC-AT compatible personal computer, using the software ASYST (Asyst Softwares Technology Inc., Rochester, NY). For experimental curves, each data point was performed in triplicate. Error bars indicate the standard deviation of experimental values. Separationswere also done three times to ensure reproducibility. It should be noted that the abscissa in all diagrams has been converted from a time scale to a distance scale.
This was done by multiplying the arrival time by the estimated average isotachophoretic velocity (u), u = d / t , where d is the distance between injector and detector and t is the time taken for the first boundary to reach the detector. Reagents. In anionic studies, HC1 (ACS reagent grade, Caledon Lab. Inc., Georgetown, Ontario, Canada) was always used as the leading electrolyte. MES [2-(N-morpholino)ethanesulfonicacid, Sigma, St. Louis, MO) was used as the tailing electrolyte. The counterions used include L-histidine (98%, Aldrich, Milwaukee, WI) or EACA (c-aminocaproicacid, Sigma). Additives, including poly(viny1 alcohol) (PVA, molecular weight 10OOO, Sigma), or poly(ethy1ene glycol) (PEG, molecular weight 200, Aldrich) were added to the leading electrolyte as a stabilizer. Solutions were prepared by using deionized water. The leading electrolyte was prepared by first dissolving the additive in deionized water. HCl was added to the desired concentration. The counterion was added last and the pH monitored by a Coming pH Meter (Model 220, Suffolk, England). Enough counterion was added to reach the correct pH, which is pH 6 for histidine and pH 4.5 for EACA. Additives and counterions were not added to the sample or tailing electrolyte. All solutions were filtered by using 0.2-pm pore size cellulose acetate filters (Sartorius,Gottingen,West Germany) and were degassed before use, by either sparging with helium or sonication. If solutions are not degassed, microbubbles form during the separation that block the passage of current and therefore hinder separation. If present, these bubbles are detected by the gradient method as bipolar signals. Samples used include citric acid (Aldrich),butyric acid (BDH, Toronto, Ontario, Canada), and acetic acid (Caledon Lab). Biodegradation products of 2 mM citric acid were prepared by incubating the solution of acid for about 48 h at mom temperature. For cationic studies, the leading electrolyte was ammonium acetate (Aldrich) and the tailing electrolyte was tetraoctylammonium bromide (TOAB, Aldrich). The samples used include vinyltriphenylphosphonium bromide (Aldrich),ethyltriphenylphosphonium bromide (Aldrich), and hexadecyltrimethylammonium bromide (Sigma). Solutions were prepared in methanol (99.970, BDH)/deionized water mixtures (50:50). Poly(ethy1eneglycol) was added to the leading and tailing electrolytes (0.05%) as a stabilizer.
RESULTS AND DISCUSSION An interesting feature of isotachophoresis is the selfsharpening effect of the zones. Pure zones of ions are formed in the initial stages of the separation, as the applied voltage drives the ions to migrate. These zones are arranged in a stacked fashion according to the descending order of their effective mobilities ( p ) from leading to tailing ion. Many factors affect the effective mobility, including absolute mobility, pK,, pH in the zone, and temperature. In a steady state, zones of ions migrate with equal velocity, ui, according to vi
=Ep
(4)
where E is the electric field gradient. Each zone of pure sample will have a specific electrophoretic mobility, and therefore, to ensure a steady velocity of the stacked assembly, E in each zone is different. This produces a self-sharpening effect. If an ion diffuses into an adjacent zone along the concentration gradient, it encounters a region of low (high) electric field strength and is decelerated (accelerated) into its own zone. This leads to a narrow boundary width (Ax) between zones that can be described by ( 4 , 5 ) (5) where R is the universal gas constant, T is the temperature (K), F is Faraday’s constant, and pwr are the mobilities of ions L and X, respectively. In eq 5, AX is defined as the distance that is required to change CL/Cx from e2 to l/e2. If we assume that the mobilities of adjacent zones are close to one another, then we can simplify the equation
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
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A where Ap is the difference in effective mobility between two analytes, neglecting the effects of zone temperatures on the mobility. Equation 6 indicates that increasing the applied potential to the separation capillary results in proportionally higher field strengths in the respective zones and causes a decrease in the boundary widths, Ax. The sharpening effect opposes diffusion and preserves sharp concentration changes at the boundaries between the zones. When the boundary, with associated high refractive index gradient, crosses the area irradiated by the focused laser beam, it deflects the beam. After the boundary has passed by, the laser beam returns to the original position. This sequence of events produces peak-shaped signals, with the maximum estimating the separation point between the two zones. Figure 3A shows a zone corresponding to a single ethyltriphenylhposphonium cation stacked between the LE, ammonium ion, and the TE, TOAB, under the usual experimental conditions and detected by the gradient sensor. The signal-to-noise ratio is excellent, exceeding 1OO:l. A further increase in the ratio can be accomplished by applying a higher potential, which will sharpen the zone as described by eq 5 and therefore increase the concentration gradients at the boundary. This increase will result in a higher sensitivity of detection. Separations illustrated in parts C and D of Figure 3 clearly demonstrate the sharpening effect. In both cases, the concentrations of analyte, LE, and T E are lowered by an factor of 10 compared to Figure 3A. This decrease causes a poor signal-to-noise ratio (SIN) (Figure 3C). This effect can be expected since lower concentrations in the zones lead to smaller differences in the refractive indices between them. The increase in applied voltage from 6 kV in Figure 3C to 10 kV in Figure 3D decreases Ax for both zones from close to 300 pm and to less than half of this value and dramatically enhances the signal-to-noise ratio of the isotachopherogram. The signal magnitude more than doubled. The decreased boundary width obtained experimentally corresponds clmely to the expected value as calculated from eq 4. The high resolution of the gradient method allows detection of the 100-pm boundaries produced in this separation. Proper detection of narrow boundaries requires use of short focal length lenses to narrow the focus spot diameter of the probing beam, which, in effect, determines the spatial resolution of the syatem. In our experimental mangement, we used a lens with 3.8-cm focal length, yielding an approximate 50-pm spot size. This diameter provides sufficient resolution to detect 300.” boundaries, but 100-pm boundaries are likely to be broadened. Decreasing the focal length of the lens yields a smaller spot size and possibly higher resolution. For example, a 10-mm lens will produce a spot size of about 10 pm. However, in order to obtain lo-” resolution, a very small inside diameter capillary needs to be used (10-50 pm) since the beam diverges very rapidly before and after the focal spot. Figure 4 shows the experimental data demonstrating the relationship between the width of the boundary ( A x ) and the applied potential. In these experiments, citric acid was used as the sample and the concentrations of all electrolytes remained constant, while the voltage applied to the separation capillary was increased from 2 to 8 kV. The theoretical asymptotic relationship, which is expected from eq 6, is indicated as a broken line. The experimental points closely follow the curve. A decrease in the boundary width occurs with an increase in the SIN of the corresponding peak. Figure 5 shows the experimental data for the same electrical potentials as in Figure 4. The S I N ratio increases dramatically for low voltages, levels off for higher values, and drops for the highest.
4.00
3.20 2.40 1 .so
.BOO
.om
z-
-.ma -1 -60
mC
0,
5
1.20
+
C
Figure 3. Capillary isotachopheretk separatlon under different experimental condltions. Sample used is ethyltriphenylphosphium cation, leading electrolyte is ammonium ion, and tailing electrolyte is tetraoctylammonkn kn. poly(ethyleneglycol) (0.05%)is added to both leading and tailing electrolytes as stabilizer. (A) 3 kV. Sample is 3 mM,LEislOmM,andTEis4.4mM. ( 8 ) 3 k V . Sampleconcentratkn is decreased compared to A. (C) 6 kV. Concentration of sample Is 0.3 mM. LE is 1.0 mM, and TE is 0.44 mM. (D) 10 kV. Concentration of sample is 0.3 mM, LE is 1.0 mM, and TE is 0.44 mM.
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
,640
:I
-.640
'
.
'
.
"
.
5 Electric Potential (kV1
'
. 1
10
Flgure 4. Boundary width versus applied electric potential across the separation caplllaty. Leading chkwide ion is 8 mM, tafflng MES is 8 mM, and citric acid sample is 2 mM. Counterion EACA, pH 4.5, is used. Poiy(viny1alcohol) (0.05%)is added to LE.
-7
I
I
30
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d d 20t z
10
"0
1
1 - ,320 - ,480
7
I
,320 .460
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Electric Potantiel [kVI
Flgure 5. Gradient detector signal height versus applied electric potentiel across the separation capi#ary,for the same conditkns as Figwe 4.
The most likely reason for the loss in S I N a t high voltages is deformation and broadening of the boundary produced by convection. This transport phenomenon is induced by density gradients generated by the temperature difference between zones (17) and the high density gradients a t the boundary. In addition, the refractive index gradients associated with the temperature gradients in the system will contribute to the overall gradient signal. However, they are expected to be broad and therefore can be easily differentiated from the sharp concentration gradient signals associated with boundaries because of several orders of magnitude higher thermal diffusivities compared to mass diffusivities (15). The effect of the thermal gradients can be only clearly seen in Figure 3D when low concentrations of the leading electrolyte and high voltages are used. Another unique property of isotachophoresis is its ability to dilute or concentrate sample components in their zones according to the concentration of the leading electrolyte and the effective mobility of the analytes in question. This concentrating effect is governed by the Kohlrausch regulating function (6)
where C is the concentration of analyte A or B, I.( is the mobility, and R is the counterion. The significance of this function is that the concentration of sample components in their zones will become proportional and close to the concentration of leading electrolyte such that the concentrations of all species (LE, sample components, TE) are constant. Thii implies that the concentrated components of the sample relative to LE will undergo a dilution effect according to the
t
c
:
-.800 -.600
Y -.400
-.ZOO
,000
.ZOO
,400
,600
,800
1 . OD
x [cml
Figure 8. Isotachopheretic separation of low concentrations of butyrate and acetate ions. Leading Ion is CI- and tailing ion is MES. Applied potential was 6 kV. Kohlrausch function. Similarly, a trace component will be concentrated until ita concentration is close to that of the leading electrolyte. The high concentrations of analytes in each zone ensure a sufficient difference in the refractive index between the zones and therefore effective detection of the boundaries between them. Even samples with quite low concentrations of analytes can be analyzed by CITP and the gradient detector. However, since mass balance must apply, as trace sample components become concentrated, the corresponding zones become very narrow. The length of the zones constitutes quantitative information. Therefore, to ensure proper determination, a high-resolution sensor, such the gradient method, is required. Figure 6 shows separation of two carboxylic acids forming submillimeter zones, comparable in dimension to the boundaries between them. In addition, the derivative nature of the detector makes it ideally suited to quantify the analytes. The location of the peak maxima clearly indicates zone dimensions. For an even lower concentration of analyte, the detection of the corresponding narrow zones becomes impossible. However, it is still feasible to perform proper quantitative determination by decreasing the leading electrolyte concentration. This will result in longer zone lengths of the lowconcentration components. This procedure is illustrated in Figure 3. Dilution of the analyte results in decreased zone length (compare Figure 3B and 3A). A 10-fold decrease in the concentration of the leading electrolyte increases the zone length substantially but results in lower signal-to-noiseratio detection of boundaries (Figure 3C). This can be improved in the case of the gradient detector by focusing the boundaries, by applying a higher electric field gradient (Figure 3D). By using this procedure, even very small amounts of ions can be analyzed. For example, to generate the isotachopherogram for Figure 3D, only a few nanomoles of sample was injected. The S f N is about 100:1, and zones are quite long. A further decrease in the leading electrolyte concentration, and focusing of the boundaries, will allow detection of a few picomoles of analyte. In addition, application of smaller internal diameter capillaries, similar to those used in capillary zone electrophoresis, will require substantially smaller volume of sample and allow application of a higher electrical potential to the system. Sharper boundaries are produced, because of better cooling, as well as reduction of convection current of solution in the small diameter capillary. The resolving power of capillary isotachophoresis is similar to capillary zone electrophoresis. According to eq 6, relative differences in mobilities between ions forming adjacent zona of only 0.001 are required to form 1-mm boundaries between them. In order to separate the same ions in capillary zone electrophoresis, the efficiency of the method should exceed a million theoretical plates. The inverse relationship between the width of the boundary and the difference in mobilities
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Anal. C b m . 1991,6 3 , 1889-1894
$500.00 and a few centimeters, respectively. The excellent compatibility of the gradient sensor and CITP indicates that this detector has the potential to be very successful in other related separation methods. For example, another electrophoretic technique that has a self-sharpening and concentrating property, capillary isoelectric focusing, involves the formation of sharp zones of analytes along a pH gradient (19, 20). In displacement chromatography, the chromatographic analogue of isotachophoresis, the samples are desorbed sequentially from the stationary phase and form sharp boundaries between them.
3.20 2.00 2,401
2.00
,000
,000
1.60
2.40
3.20
4.00
4.00
5.60
6.40
7.20
x Icml
Flgure 7. Isotachopherogramof bbdegradatbn mixture. Chloride ion (LE) and MES (E) were at 8 mM. Applied potential was 3 kV.
of ions, results in a trade-off between resolution of CITP and the detection limits for the concentration gradient detector. Sharp zones, which are required for low level detection of ions, are formed only when ions differ substantially in mobilities. A major advantage of CITP over zone electrophoresis is the upper diameter limit of the capillary, which is required for successful separation. One-millimeter inner diameter capillaries can be effectively used as shown in Figures 3 and 6. Therefore, CITP is more suitable for micropreparativework, for example, to obtain high-purity biological materials (8). Figure 7 illustrates such an application where small concentrations of two biodegradation products are separated out from the main sample citric acid. The two zones of the products follow the citric acid, forming in total three zones between the LE and the TE. The gradient detector is very useful in such an application by indicating the arrival of the zone of interest for collection. Smaller inner diameter capillaries can also be used for analytical-scale, high-resolution separation. In this case, the effect of broadening due to the convection generated by high-density gradients is small. The only qualitative information provided by isotachophoresis is the elution order of a given analyte, which corresponds to its mobility compared to other ions present in the mixture. The gradient detector provides information about the refractive index of the zones. This can be accomplished by integrating the gradient response, as it is shown in Figure 1D. It can also provide spectroscopic adsorption information about analytes by using a selective mode of the gradient method (18). However, to characterize unknown samples, mass spectrometryshould be applied (7). CITP provides pure zones for such determinations,which eliminates interferences and simplifies qualitative analysis. The remote and nondestructive nature of the gradient detector is very useful in such schemes since it marks the zones without interfering with the analysis. The cost and size of the detection system is small, particularly when laser diodes are applied as the light source. The ease of focusing is similar to He-Ne lasers when visible laser diodes, and widely available collimate optics, are used. The sensitivity of detection is similar to that of the He-Ne laser, but the cost and size of the system is cut substantially to about
ACKNOWLEDGMENT Beckman Instruments Inc. provided the high-voltage power supply. Registry No. Citric acid, 77-92-9; butyric acid, 107-92-6;acetic acid, 64-19-7; vinyltriphenylphosphonium bromide, 5044-52-0; ethyltriphenylphosphoniumbromide, 1530-32-1; hexadecyltrimethylammonium bromide, 57-09-0.
LITERATURE CITED Jorgenson. James; Philps, Marshal. New Dbctbns in €bm#wetiC Mefhods ; ACS Symwslum Series 335;Amerlcan Chemical socclety: Washington, DC, 1987. Wailingford, Ross A.; Ewlng, Andrew 0. In Advances In chrometogrephy; Glddlngs, Calvin, Grushka, Eli, Brown, Phyllls R., Eds.; Marcel Dekker Inc.: New York, 1989 Vol. 29, pp 1-76. Bocek, Pew Deml, Mlrko; Gebauer, Pew Dolnlk, Vladislav. A n a w l Isotechophores~ ; VCH Publishers: Weinhelm, West Germany, 1988. Everaerts, Frans; Beckers, Jo: Verheggen, b o . P. E. M. Isofachophoresls; Elsevier: New York, 1976. Everaerts, Frans, Ed. Anaiytkxl Isotachophoreskr , Proceedings of the 2nd Internatbnal Symposium on Isotachophoresls; Eisevisr: Amsterdam, Oxford, and New Yo&, 1981;pp 1-234. H!almarsson, Sven G.;Baldesten, Astor. CRC &it. Rev. Anal. Chem. 1981, 11 (4),264-352. Smith, Rlchard S.;LOO, Joseph A.; Edmonds, Charles 0.; Barinage, Charles J.; Wseth, Harold R. Anal. Chem. 1989. 61. 228-232. Firestone, Millicent A.; Micheud, Jon Pierre; Carter, Richard H.; ThorITtann, Wolfgang. J . chroma*. 1987, 407. 363-368. Konstantlnov, 8. P.; Oshurkova, 0. V. Sov. Fhys.-Tech. Phys. (Engl. Transl.) 1986, 1 1 , 693-704. Bruno, Alfredo E.; Ciessmann. Ernst; Pericles, Nico; Anton, Klaus. Anal. Chem. 1989. 61. 876883. Pawllszyn. Janusz. Spectrochem. Acta Rev. 1990, 13 (4),311-354. Pawllszyn, Janusr. Anal. Chem. 1988,58, 3207-3215. Pawllszyn, Janusr. J . Uq. Chromatop. 1987. 10, 3377-3392. Pawllszyn, Janusz. Anal. Chem. 1988,6 0 , 2796-2801. McDonnell, Theresa; Pawllszyn, Janusz. Capillary Isotachophoresls with Concentration Gradient Detection: An Appliltion to the Separation of Synthetic Peptides. J . Chromalop., In press. Pawiiszyn, Janusz. Rev. Sci. Insf”. 1987,58 (2),245-248. Reijenga, J. C.; Verheggen, Th. P. E. M.; Everaerts, F. M. J . Chromatop. 1985,328,353-356. Pawliszyn, Janusz. Anal. Chem. 1988, 60. 766-773. Hjerten, Stellan; Zhu, Mlng-De. J . Chromatog. 1985,346, 265-270. Hjerten, Stellan; b o , JaN; Yao, Kunquan. J . Chrometogr. 1987,
387,127-136.
Theresa McDonnell Janusz Pawliszyn* The Guelph-Waterloo Centre for Graduate Work in Chemistry University of Waterloo Waterloo, Ontario, Canada N2L 3G1
RECEIVED for review November 19,1990.Accepted May 29, 1991. This work was supported by the National Science and Research Council of Canada and Eward Bioengineering.
Ion Chromatographic Separations Using Step and Linear Voltage Waveforms at a Charge-Controllable Polymeric Stationary Phase Sir: Separations using ion chromatography generally rely on a stationary phase with a fixed composition, Le., a fixed number of exchange sites (1,2).With such phases, changes 0003-2700/91/0363-1889$02.50/0
in separation characteristics are accomplished by altering the composition and flow rate of the mobile phase. Recently, the possibility of manipulating separations via electrochemically 0 1991 Amerlcan Chemical Society
