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Anal. Chem. 1907, 5 9 , 472-476
Column Efficiency in Micellar Electrokinetic Capillary Chromatography Michael J. Sepaniak* a n d Roderic 0.Cole
Department of Chemistry, University of Tennessee, Knoxuille, Tennessee 37996-1600
Expertmental factors that influence column efflclency in micellar eiectroktnetic capillary chromatography were studied. Parameters, such as anpUed voltage, cokrmn dkmmslons,and concentrationsof buffer and surfactant In the mobile phase, are found to influence efflclency. Van Deemter-like plots of plate height vs. applled voltage are used to demonstrate the slgnlflcance of factors which typlcaHy cause band dlsperslon in chromatography and electrophoresis. Msperslon due to resistance to mass transfer In the mobile phase and temperature gradlents wlthin the column are shown to be most slgnlflcant. Plate helghts of less than 10 pm are possible when experimental parameters are optimized.
Capillary zone electrophoresis (CZE) is an efficient separation technique in which charged solutes are differentially transported through open capillaries under the influence of an applied field (I). The capillaries efficiently dissipate heat generated by the electrophoretic process, and consequently, high voltages can be applied in CZE without thermal problems. Moreover, electrokinetically pumped capillaries have pluglike flow profiles that exhibit better mobile-phase mass transfer characteristics than hydrostatically pumped capillaries, which have parabolic flow profiles (2). Efficiency in CZE is limited primarily by axial diffusion. Rapid transport through the capillary at high applied voltages minimizes solute band dispersion due to axial diffusion. The result is high separation efficiency, often exceeding 100000 plates/m. Despite the high efficiency of the technique, CZE is not very effective at separating neutral solutes, since they are transported through the capillary strictly by electroosmotic flow (3) and have retention times that differ only slightly; i.e., separation selectivity is poor for neutrals. Two approaches have been directed at extending the CZE technique to neutrals. The first involves adding a large tetraalkylammonium ion to the mobile phase, which imparts a charge to neutral solutes via solvophobic interactions (4). The resulting charged species can be separated electrophoretically. The second, more widely used approach, which we have termed micellar electrokinetic capillary chromatography (MECC), involves the addition of surfactant ions to the mobile phase a t concentrations above their critical micelle concentration (cmc)(5). Neutral compounds are separated based on their differential partitioning between an electroosmotically pumped aqueous mobile phase and the hydrophobic interior of the micelles, which are moving at a velocity different from that of the mobile phase due to electrophoretic effects. Ions sorbed onto the inside surface of a glass capillary column generate an electric double layer potential ({potential). The magnitude and sign of the { potential influence the magnitude and direction of electroosmotic flow. In our work with acid-washed fused-silica capillaries, using both anionic (e.g., sodium dodecyl sulfate, SDS) and cationic (e.g., cetyltrimethylammonium chloride, CTAC) surfactants, the electroosmotic flow opposes the electrophoretic flow of the micelles and is of a greater magnitude. Consequently, two distinct
phases, aqueous (mobile) and micellar (pseudostationary), exist within the column and migrate a t different velocities toward the electrode with the same charge as the micelles. Solutes injected into the inlet end of the capillary are eluted with retention times which range from to,the retention time of a solute which is not solubilized by the micelles, to t,, the retention time of a solute which is totally solubilized (5). Although the MECC technique can provide for the efficient separation of neutral compounds, they must have reasonable solubility in the aqueous mobile phase. Nevertheless, the technique has been used for the separation of a variety of samples including phenolic compounds (6) and other substituted benzenes (5, 7), phenylthiohydantoin amino acids (B), metabolites of vitamin B6 (9), nitrated polyaromatic hydrocarbons (51, and purines (5,101. Fundamental characteristics of MECC that influence retention behavior have been investigated (11). However, except for a report of the effects of injection procedures (lo), experimental factors that influence column efficiency in MECC have not been studied. This report concerns our investigations into the effects of experimental parameters, such as applied voltage, column dimensions, and concentrations of the buffer and surfactant in the mobile phase, on column efficiency in MECC. When the results are presented in the form of Van Deemter-like plots of plate height vs. applied voltage, it can be seen that very high efficiency is possible, but only if close attention is given to experimental conditions. The data from these investigations are used in a qualitative discussion of the importance in MECC of factors which traditionally contribute to solute band dispersion in chromatography and electrophoresis. EXPERIMENTAL SECTION Apparatus. The basic experimental arrangement consisted of a length of fused silica capillary with each end immersed in mobile-phase reservoirs maintained at equal heights to prevent hydrostatic flow. A regulated high-voltage dc power supply, delivering 0-40 kV (Hipotronics, Inc.), was used to provide the electric field necessary for electrokinetic flow. On-column fluorescence detection was preformed by using an-argon ion laser (Cyronics Model 2001-20BL) for excitation. The laser was operated at a power of 20 mW and a wavelength of 488 nm. Fluorescence emission at 540 nm was collected (at right angles relative to the incident beam) and isolated with an SA Instruments, Inc., Model H-10 monochromator. The monochromator was set to 540 nm with a 6-nmband-pass. Signals were monitored with an RCA Model 1P28 photomultiplier and processed with a Pacific Precision Instruments Model 126 quantum photometer. Two 25 mm X 35 mm achromat lenses (Oriel Corp.) were used to focus the laser radiation onto the flow cell and for the collection of fluorescence. Optical windows for detection were made by heating a short section of the polyimide coating on the fused silica near one end of the capillary. The coating oxidizes readily and can be removed with a lens cloth to form a window. A plastic collar was then attacked just above the window with epoxy and affixed in a fiber-optic positioner (Newport Corp., Model FP-1). Alignment of the flow-cell window was accomplished by noting the shape of the far-field images. When aligned, the image of the incident beam directly behind the column assumes an oval shape. The image of the transmitted beam is focused with the collection lens onto the emission monochromator slit plate and appears as
0003-2700/87/0359-0472$01.50/0 0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
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Table I. Column Factors Contributing to Observed Plate Height in MECC
factor
H A axial diffusion Hs resistance to mass transfer,
V(l), Nt),R ( t ) Ut), R ( t ) ,s(t)
HM,,, resistance to mass transfer,
Wt), Wl), R(l), [ml(l)
pseudo stationary phase
RETENTION TIME ( M I N , )
Figure 1. MECC chromatogram of NBD-ethylamine (A) and NBD-
cyclohexylamine (B). Separation and detection conditions are given in the text. four equally spaced lines, each line resulting from specular scatter at the inside and outside interfaces of the column. The monochromator is positioned such that the specular scatter is rejected by the slit plate while the fluorescence eminating from the center of the capillary is passed by the slit. Reagents and Materials. Columns. Capillary columns of 25,75, and 100 fim i.d and lengths of 75 cm were employed. The columns were washed with 0.1 M HC1 and then filled with the mobile phase. The mobile phase used in all experiments, except the micelle concentration and thermal dispersion studies, was composed of 0.003 M disodium phosphate and 0.01 M SDS in distilled,deionized water. Solutions were initially introduced into the capillaries by attaching a steel fitting to one end with epoxy and applying gas pressure to a liquid reservoir containing the desired solution. Subsequently, the mobile phase can be rapidly changed by simply changing the buffer solution reservoirs. No column equilibration was necessary. Electroinjection. Injections were accomplished by replacing the mobile-phase reservoir with a sample container and applying a potential of 1 kV for 15 s. Longer times introduce too large a zone into the column, causing low efficiency and poor peak shape (10).
Measurement of Peak Efficiency. Peak efficiencies were calculated by using the relationship 2
H=-("> L 5.54
t,
where H i s height equivalent to a theoretical plate, L is length of column, W is peak width at half maximum in time units, and t, is retention time. Reagents. The buffers and other chemicals were of reagent grade or better. SDS was obtained from Sigma. All amines and 4-chloro-7-nitrobenzofurazan(NEiD-Cl) were obtained from Fluka Chemicals (Hauppauge, NY). Amines were derivitized with NBD-C1 as described by Sepaniak and Murray (12).
RESULTS AND DISCUSSION A sample chromatogram for the separation of the test solutes used in this work is shown in Figure l. The column was 75 pm i.d. X 75 cm and the applied voltage and resulting current were 15 kV and 10 MA,respectively. Since we did not include an extremely hydrophobic compound in the mixture to mark t,, it is not possible to calculate retention parameters for the test solutes (11). However, our previous experiences with the technique under these conditions resulted in values of to/t, of about 0.4. Hence, the retention ratio (R),Le., the fraction of solute which is not solubilized by micelles, of NBD-cyclohexylamine is roughly a factor of 5 smaller than that of the NBD-ethylamine. There are several kinetic processes which serve to disperse solute bands as they are transported through a chromatographic column. The influences of these processes on column plate height are additive. Extracolumn processes (principally injection and detection) can also cause band dispersion. In this work, on-column electroinjection (10) and on-column laser-excited fluorescence detection (13) were performed. Hence, extracolumn band dispersion is expected to be neg-
influence of exptl parameters" (effect of increasing parameter)
mobile phase (intermicelle) HM,, resistance to mass transfer, V(t), D(l), Nl),d,2(t) mobile phase (intracolumn) HT thermal dispersion P I U t ) , dAt) Experimental parameters: V, applied voltage; D, diffusion coefficient; R, retention ratio; S, micelle size; d,, column diameter; [ m ] micelle , concentration; P/L,power/length. ligible. The total plate height (H)for MECC is then given by eq 2, where the individual terms are defined in Table I.
Since MECC separations are performed in open capillaries, eddy diffusion is not problematic. This accounta for the absence of the term in eq 2. Predictions are given in Table I concerning the qualitative effects (increase t or decrease J) that experiment parameters will have on the individual contributions to H. These predictions are inferred from existing theoretical descriptions of solute band dispersion in chromatography and electrophoresis. Exact expressions are difficult to derive for MECC due to the complication of having two moving phases. For example, an expression for the plate height contribution due to axial diffusion (HA)in conventional open capillary chromatography can be derived in a straightforward manner from Einstein's law of diffusion. In that case HA is simply twice the diffusion coefficient (D)of the solute in the mobile phase divided by the linear velocity of the mobile phase (14). Since all solutes, regardless of their retention, spend the same length of time in the mobile phase, there is no dependence on a retention parameter. In MECC all solutes do not spend the same length of time in the mobile phase. Ignoring axial diffusion of the micelle solubilized solute, the more complicated MECC expression for HA is
(3) where Ve,is the electroosmotic flow velocity and V,, is the electrophoretic flow velocity of the micelles. Equation 3 indicates that as diffusion coefficient and retention ratio increase, plate height increases and efficiency degrades. Because of the direct relationship between applied voltage and the velocity terms appearing in the denominator of eq 3, increases in voltage decrease HA.This is illustrated in Figures 2-4, which are plots of plate height vs. applied voltage under various experimental conditions. At low voltages H A is significiant and increases in voltage reduce the observed plate height. The plots for MECC contrast those for CZE in that sources of band dispersion other than axial diffusion (see the discussion below) become important at relatively low voltages. The value of Veoa t 15 kV for this system is approximately 0.1 cm/s. At high voltages, the plots in Figures 2-4 are similar to conventional chromatography plots in that they show a direct relationship between plate height and applied voltage. This indicates the significance of dispersion due to resistance to mass transfer, which is generally true at high mobile-phase velocities. Figure 2 suggests that resistance to mass transfer in the pseudostationary phase (H,) is not important since the
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
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40
35
25-
30
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,E..
E
x
3.
c
t-
0
I
ww =z w
25
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15-
4
a
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VOLTAGE ( K V )
Flgure 2. Plate height vs. applied voltage plots for NBD-cyclohexylamine (A) and NBD-ethylamlne (B). A 75-pm4.d. by 75-cm column filled with a 0.01 M SDS, 0.003 M Na2HP04sdutbn was used to obtain the data for the plots.
I
I
I
I
10
20
30
40
VOLTAGE (KV)
Figure 4. Plate height vs. applied voltage plots for NBD-cyclohexylamine obtained by using a high (0.007 M Na,HPo,) buffer concentration (A) and a low (0.003 M Na,HPO,) buffer concentration (B). Other conditions were the same as used for Figure 2. Table 11. Effect of Surfactant Concentration on Efficiency NBD-ethylamine [SDS],M 1.0x 5.0 x 1.0 x 2.0 x 5.0 X
10-3 10-3 10-2 10-2
NBDcyclohexylamine TR,min N
current, pA
TR,min
14 15
4.0 5.0
20 32
6.3
6 900n 39000
7.4 8.5
72000
10.4
7 900 17000 30000
65000
10.7
37000
59
N
4.0 6.6 9.9
NBD-ethylamine peak and solvent disturbance only partially resolved.
SDS micelles have radii of about 20 A) should render the Hs term insignificant. However, we have encountered situations
I
10
20
30
I
40
VOLTAGE ( K V )
Flgure 3. Plate height vs. applied voltage plots for NBD-cyclohexyiamine obtained by using
[email protected]. (A) and 25-pm4.d. (B) columns. Other conditions were the same as used for Figure 2. high-voltage slope of the NBD-ethylamine plot is less than that of the NBD-cyclohexylamine plot. The former solute has a retention ratio ( R )which is several times larger, and as indicated in Table I, this should result in a larger, rather than smaller, slope for the NBD-ethylamine, providing Hs is significant. In fact, the theoretical expression for Hs in conventional chromatography shows only a weak dependence of Hson R (14). Furthermore, micelle size should be analogous to stationary-phase thickness in conventional partition chromatography, and the small dimensions of micelles (e.g.,
when strong micelle-solute electrostatic interactions appear to have resulted in poor stationary-phase mass transfer kinetics. For example, the separation of a mixture of substituted benzene compounds by using a cationic surfactant yielded relatively poor efficiency for the weakly acidic phenol (5). While MECC separations are conducted in open capillaries, the columns can behave as packed columns with the micelles functioning as uniformly sized and evenly dispersed packing particles. In packed columns, resistance to mass transfer in the mobile phase is reduced when smaller particles are employed. With smaller particles the “diffusion distance” between particles is decreased. Smaller interparticle diffusion distances improve the kinetics of mobile-phase mass transfer. Average “intermicellar distance”, the analogous MECC parameter, can be decreased by increasing surfactant concentration. The results of a study of the effect of surfactant concentration on efficiency for a particular MECC system are shown in Table 11. A
[email protected] was used in that study. The cmc for SDS is approximately 8 X M. However, impurities in the SDS reagent and the high ionic strength of the mobile phase can alter the concentration at which micelles are formed. The data in Table I1 indicate some retention
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
occurs a t an SDS concentration of 5.0 X M. Retention time increases as the SDS, hence micelle, concentration is increased. The NBD-cyclohexylamine elutes near t, when the micelle concentration is large. This is reflected in the small changes in t, when the micelle concentration is changed. The effect of increasing micelle concentration on efficiency is dramatic, particularly at low micelle concentrations. One possible explanation for the large improvement in efficiency as micelle concentration is increased is that “intermicelle” resistance to mass transfer (HM,,) is a significant factor in the total plate height and it is reduced as intermicelle distance is reduced. Assuming a cmc of 8 x M and an aggregation number of 62, the average intermicellar distance for a 5.0 X M micelle solution is only about a few hundred angstroms. This distance is so small when compared to the interparticle spaces in a conventional packed columns that it would seem that HM,, should be insignificant even at very low micelle concentrations. However, the high efficiency of the MECC technique can add significance to otherwise negligible sources of band dispersion. Furthermore, the magnitude of HMsnwill depend on the mobile-phase flow profile between the micelles, which to our knowledge has never been studied. The reduction in efficiency (shown in Table 11) for the highest SDS concentration is due to thermal-related dispersion and is discussed later. When separations are performed with true open capillaries, which as discussed above is not strictly the case in MECC, the major source of band dispersion is generally “intracolumn” resistance to mass transfer ( H M , ~ The ) . transverse velocity profile of the mobile phase is parabolic in hydrostatically pumped capillaries. Dispersion occurs since solutes near the center of the capillary are moving faster than those located near the stationary phase a t the walls of the capillary. The dispersion is less for the pluglike flow profiles of electrokinetically pumped capillaries; however, HM,c can still be significant (see below). If a solute can diffuse rapidly across the velocity profile, the dispersion is minimized. Large diffusion coefficients and extremely narrow-bore capillaries are thus desirable. Figure 3 is a comparison of the efficiency of the NBDcyclohexylamine peak as a function of applied voltage for 75-pm-i.d. (A) and 25-pm4.d. columns (B). By use of a 0.003 M disodium phosphate buffer, the current was kept low enough to prevent thermal problems. At high voltages, the condition for which resistance to mass transfer is expected to be important, the small inside diameter column exhibits much better efficiency, indicating that H M ,is~a very significant source of dispersion. The high-voltage slope of the plot for the 75-p-i.d. column is roughly a factor of 5 greater than for the 25-pm-i.d. column. The fact that the theoretical quadratic dependence of H M ,on ~ column diameter (14) is not observed could possibly be attributed to the fact that plate height is plotted vs. applied voltage and not mobile-phase velocity. Moreover, the study was conducted using a SDS concentration of 0.01 M. Information from Table II indicates the significance of H M , ~which , should be independent of column diameter, at this surfactant concentration. Figure 2 illustrates the importance of diffusional processes in determining efficiency in MECC and further indicates the significance of Hwc. The figure contains the Van Deemter-like plots for NBD-cyclohexylamine (A) and NBD-ethylamine (B). These solutes might be expected to have similar diffusion coefficients in the aqueous mobile phase. Both HMm and should show an inverse dependence on diffusion coefficient. However, the former should depend on the true solute diffusion coefficient in the aqueous mobile phase while the latter should depend on the “observed” diffusion coefficient in the mobile phase (15). The NBD-cyclohexylamine might then
475
be expected to exhibit much greater intracolumn resistance to mass transfer in the mobile phase, since it spends much more time in the bulky micelle (i.e., has a much smaller observed diffusion coefficient). This is consistent with the high-voltage slopes in the figure. A t low voltages, where HA should be the most significant source of dispersion, the ethylamine derivative plot should have a greater negative slope. Unfortunately, there are not sufficient data at low voltages to draw any firm conclusions concerning this from Figure 2. An advantage of CZE over other forms of electrophoresis is the ability of narrow-bore capillaries to limit the electrophoretic thermal load (16)and to efficiently dissipate heat. Nevertheless, when the electric power dissipated per unit column length is too large (i.e., when voltages and currents are too large) a transverse temperature gradient is created within the capillary. Under these conditions the mobile-phase temperature is greater in the center of the capillary than it is near the walls where the heat is conducted to the environment. In general, narrowing the capillary bore lessens the temperature gradient. Since electrophoretic mobility increases with temperature, charged solutes which are distributed across the diameter of a capillary assume a dispersive velocity profile, when a temperature gradient exists. It is reasonable to expect the pluglike velocity profile of electroosmotic flow to be distorted similarly, and this should increase the dispersion of neutral solute bands in MECC (i.e., in effect, HM,c will increase). Another thermal dispersion effect that can occur in MECC, but not in CZE, relates to the temperature dependence of cmc values. It has been shown that the cmc of SDS increases from approximately 8.4 X M at 25 OC to approximately 9.8 X M at 55 “C (17). The surface temperatures of MECC capillaries depend on the power dissipated and are typically tens of degrees Celsius above ambient temperature (11). This indicates that for a constant SDS concentration increasing the power dissipated in the capillary will increase the cmc, decrease the micelle concentration, and increase H M m due to a concomitant increase in intermicelle diffusion distance (see previous discussion). When surfactant concentrations near the cmc are used, this may be an important factor in determining efficiency. The limiting power per unit length for the capillaries used in this work is roughly 2 W/m. Higher values often resulted in boiling of the mobile phase, at which point the current ceased and the column had to be refilled with mobile phase. Even at lower powers the thermal dispersion problems discussed above can be significant. This is illustrated by the Van Deemter-like plots for NBD-cyclohexylamine in Figure 4. Plot B is the same as those appearing in Figures 2 and 3. However, plot A was obtained by using a higher (0.007 M) buffer concentration. For a given capillary and voltage, a major effect of increasing the ionic strength of the mobile phase is to increase the current and hence the power dissipated. The last data point in plot A was at 32.5 kV and 60 pA while the last data point in plot B was at 35 kV and 2 1 pA. The mobile phase with the higher buffer concentration exhibited better efficiency at intermediate voltages. However, at high voltages the current is excessive as reflected in the very rapid degradation in efficiency as voltage is increased. The data in Table I1 also illustrate the importance of limiting the power dissipated in the capillary. The applied voltage was 20 kV for this experiment. The general trend is for efficiency to improve as SDS concentration is increased due to a reduction in intermicelle distance. The trend changes at the highest SDS concentrations, presumably due to high current and the related thermal-related dispersion. It is interesting to note that the thermal dispersion appeared to be
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
more severe for the NBD-ethylamine, which is poorly solubilized by the micelles, then for the well-solubilized NBDcyclohexylamine. A possible explanation for this is that a temperature gradient in the capillary causes a distortion in the pluglike electrophoretic velocity profile of the micelles, which are attempting to migrate in the opposite direction as the electroosmotic flow, and this tends to compensate for a similar distortion in the electroosmotic flow profile. This compensation would be greatest for solutes that are reasonably solubilized by the micelles.
CONCLUSION MECC possesses many advantages relative to other analytical separation techniques. It can be used to effectively separate neutral compounds, whereas CZE is primarily restricted to ionic compounds. The MECC technique is much more efficient than conventional liquid chromatography (LC). We have routinely obtained efficiencies in excess of 100 000 plates/m for rapid separations. In comparison to true open capillary LC, it is not necessary to undertake involved procedures to bond stationary phases to the capillary walls, which often result in column occulsion. We have found the MECC technique experimentally simple to use. Moreover, excellent efficiency is possible by using relatively large capillaries, thereby reducing injection and detection problems relative to open capillary LC. The MECC technique is in a developing stage and its ultimate analytical utility remains to be determined. The limited elution range of the technique and the need for moderate solute solubility in the aqueous mobile phases which have been employed thusfar, are restrictions of the technique which must be addressed. Nevertheless, preliminary reports have demonstrated potential for MECC. In this paper we have reported our observations concerning the influence of various experimentally controllable parameters on chromatographic efficiency. Facton that influence efficiency in MECC are complex and this necessitates strict attention to experimental details if high efficiency is to be attained. In summary, a t voltages that yield rapid separations, resistance to mass transfer in the mobile phase, intermicelle (IfMm) and intracolumn (HM,J,appear to be the primary sources of band dispersion. High surfactant concentrations
and small capillary inside diameters (but not as small as are necessary for high efficiency in open capillary LC) are important in minimizing H M and ~ Hwc,respectively. Care must be taken not to increase voltage or the ionic strength of the mobile phase to the point where the thermal load on the capillary is excessive, producing band dispersion. In this work power dissipations of less than 1.0 W/m resulted in efficient operation. The results presented herein were for a single, simple test mixture and a particular MECC system. The same experimental parameters are likely to be important in optimizing efficiency for other systems; however, optimum conditions may vary considerably from those demonstrated in this report.
LITERATURE CITED (1) Jorgenson, J. W.; Lukacs, K. D. Anal. Cbem. 1981, 53, 1298-1302. (2) Martin, M.; Gubchbn, G. Anal. Chem. 1984, 56, 614-620. (3) Pretorius, V.; Hopkins, B. J.; Schleke, J. D. J . Cbromatogr. 1974, 99, 23-30. (4) Walbrochl, Y.; Jorgenson, J. W. Anal. Cbem. 1986. 58, 479-481. (5) Sepaniak, M. J.; Burton, D. E.; Maskarinec, M. P. Ro#edings of Symposlum on The Use of oldered Medla In Separations, ACS Symphosiurn Series American Chemical Society: Washington, DC, in press. (6) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Cbem. 1984, 56, 111-113. (7) Terabe. S.; Ozaki. H.; Otsuka, K.; Ando, T. J . Chromafogr. 1985, 332.211-217. (8) Otsuka, K.; Terabe, S.; Ando, T. J . Cbfomafogr. 1985, 332, 219-226. (9) Burton, D. E. Sepaniak, M. J.; Maskarinec, M. P. Cbromafogr. Sci. 1986, 24, 347-351. (10) Burton, D. E.; Sepaniak, M. J.; Maskarinec, M. P. Chromatographia 1988, 21, 583-586. (11) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834-841. (12) Murray, G. M.; Sepaniak, M. J. J . Ll9. Cbromatogr. 1983, 6 , 931-937. (13) Sepaniak, M. J.; Vargo, J. D.; Kettler. C. N.; Maskarlnec, M. P. Anal. Cbem. 1984, 56, 1252-1256. (14) Giddings, J. C. Dynamks of Chromatography; Marcel Dekker: New York, 1965. (15) Armstrong, D. W.; Ward, T. J.; Berthcd, A. Anal. Cbem. 1988, 58, 579-582. (16) Lukacs, K. D.; Jorgenson, J. W. HffC CC,J . High ffesolut. Chromatogr Cbromafogr . Commun . 1985, 8 , 407-4 11. (17) Goddard, E. D.; Benson, G. C. Can. J . Cbem. 1957, 35, 986-991.
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RECEIVED for review July 29,1986. Accepted October 1,1986. This research was sponsored by the Division of Chemical Sciences, Office of Basic Energy Sciences, US.Department of Energy, under Contract DE-ASO-83ER13127 with the University of Tennessee (Knoxville).
