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ANALYSIS OF FACTORS ZONE ELECTROPHORESIS

CAUSING

PEAK

BROADENING

IN CAPILLARY

XIAOHUA

HUANG,

WILLIAM

F. COLEMAN”

and RICHARD

N. ZARE*

Department qf Chemistry, Stanford University, Stanford, CA 94305 (U.S.A.)

SUMMARY

An equation analogous to the Van Deemter equation in chromatography is developed to account for peak broadening in capillary zone electrophoresis (CZE). This equation applies to conditions where the peaks are symmetrical (sample zone concentration much less than background electrolyte concentration). To identify and to quantitate the effects of different contributors to the peak width in CZE, it is first necessary to put all peak profiles on the same footing by correcting them for the velocities of different zones and for the finite length of the detector zone compared to the sample zone. Three major contributors to the peak width are identified: (1) the injection length of the sample; (2) longitudinal diffusion that takes place during the migration time between injection and detection; and (3) analyteewall interactions. Temperature is shown not to be a major factor in peak broadening under typical experimental conditions. The predictions of our model agree well with experimentally determined peak profiles for different analytes under a variety of conditions. New expressions for theoretical plate number and resolution in CZE are presented. It is concluded that in almost all previously reported CZE separations the peak profiles were dominated by the sample injection length. This explains why the-observed peak widths in CZE have been broader than anticipated.

INTRODUCTION

As the use of capillary zone electrophoresis (CZE) for separation and quantitation becomes more common1-4, it is increasingly important to understand those factors that control the experimentally observed peak widths. Without this knowledge, it is not possible to establish the optimum design parameters for a CZE separation and to assess the efficacy of this new separation method compared to others. Several models have been developed to account for peak broadening in chromatographic separations. The simplest and most commonly used of these is the so-called Van Deemter equation5, which has been extensively tested by Katz et ~1.~ and Jones7. We develop a similar equation for modeling peak broadening in CZE. a Permanent

0021-9673/89/$03.50 address: 0 Department of Chemistry, Wellesley College, Wellesley, MA 02181, U.S.A.

1989 Elsevier Science Publishers

B.V

This requires another correction before the peak widths can be analyzed in terms of broadening mechanisms. Brewster. as well as Grushka et al. Hipotronics.) in all of these studies had inside diameters of 75 pm. Japan Spectroscopic Co. Both hydrostatic and electrokinetic injection were used. Because we can model peak broadening in CZE accurately. Japan) modified for CZE detection. and C = capacitance of circuit) of the system. the model is able to predict successfully the observed dependence of peak width on migration time and injection length. The latter authors concluded that longitudinal diffusion is the dominant broadening mechanism at low velocities. observed peak widths do not directly reflect the actual spreading of the zone in the migration channel unless an appropriate correction is applied. In each case.S. ZARE There have been several previous studies of zone broadening in CZE. Fig. we model them in terms of intrinsic contributions from diffusion and analyte-wall interactions along with an extrinsic contribution from the length of the injected sample plug. This model is tested against experimental data for several different species: a positively charged. F. and voltages of 2-30 kV were applied. primarily with micellar solutions. Because of the different residence times in the detector region. for capillary dimensions of 100 pm I.A. if desired. W. The capilllaries (Polymicro Technology. AZ. N. a neutral. The high-voltage circuitry (R50B HV power supply.S. This minimized the rise time and fall time of the injection voltage when electrokinetic injection was used. the distance from the injection point to the detector was between 40 and 120 cm. R. we show that peak width measurements may be used to estimate either the diffusion constant or the length of the injected plug. 1 shows the injection current obtained with and without the modification to the injection circuit described here.) was modified by the addition of a 80 MQ load resistor to decrease the RC time constant (where R = resistance of electrolyte inside the capillary. Separations by CZE have many of the characteristics of traditional chromatographic separations but also differ in some important aspects. Once peak widths have been properly corrected for these two effects. and a negatively charged analyte.” on zone broadening in the electrokinetic chromatography of uncharged solutes. Additionally. Phoenix. while analyte-wall interactions (sorption-desorption kinetics) and heterogeneity become significant factors at higher velocities. the detector zone in CZE can have a length that is not negligible compared to the sample zone width. and Knox and Grantg. There have been related studies by Sepaniak and Colel’ as well as by Terabe et al. U. or less. U. lo have considered at length the effect of temperature gradients. NY. Hjerten*. Unlike chromatographic separations. EXPERIMENTAL The experimental apparatus used in this work has been described previously13. COLEMAN. This is an important consideration when quantifying the size and shape of the injection plug.A. different zones in CZE move past the (on-column) detector at different velocities. However.96 X. as is typical of most CZE separations. Detection was accomplished either by laser-induced fluorescence (LIF)i4 or by absorption using a high-performance liquid chromatography UV spectrophotometer (UVIDEC-100-V. the effect of heat generated within the capillary tube on the separation efficiency is rather unimportant.. HUANG. and for currents of 30 PA or less. Under certain conditions.D. Tokyo. . we can design separations that have improved resolution.

to have symmetrical profiles. In almost all of the experiments. 20 mM sodium phosphate at pH 7. retention times arise from a complex series of interactions between sample molecules in the mobile phase and the components of the stationary phase and from mobile phase-solute interactions. 1. also affect the shapes of the chromatographic peaks. the buffer concentration was at least 400 times greater than the sample concentration.) and were used without further purification. consisting ideally of a single component of the analyte mixture surrounded by buffer. This flow rate depends on the back pressure of the mobile phase and any other applied driving force. The data are recorded using analog-to-digital conversion with the help of an IBM PC/XT computer. Plot of injection current vs. To check this possibility. Because the conductivity of the sample zones is essentially equal to that of the column as a whole. The peaks were then expanded up to 60 times in width and were found. giving a time resolution of better than 0. MO. to the All of the compounds studied were obtained from Sigma (St. Thus. Such non-uniform field effects have already as been reported in CZE 8. RESULTS Correction for zone velocity In separation methods such as liquid and gas-liquid chromatography. moves at a velocity that is the vector sum of the electrophoretic velocity of that component and the electroosmotic velocity of the bulk solution under the conditions of a given experiment. U. namely. .16 . Louis.A.15. Irrespective of the peak broadening mechanisms that are operative. time for electrokinetic capillary (dashed line) and with an 80 MO load resistor injection without a load resistor in parallel (solid line) in parallel to the capillary. in all cases. A zone. These effects lead to asymmetric peak broadening well as peak narrowing caused by the fact that the leading edge of a zone migrates at a different rate than the trailing edge.1 s. Samples were prepared in a buffer identical to that used as the supporting electrolyte. effects arising from non-uniform electric fields are minimized. the peak profiles were sampled at 15 Hz.PEAK BROADENING IN CZE 97 0 4 6 8 10 TIME (s) Fig. It is generally accepted that these interactions.S. The situation is markedly different in CZE. together with diffusion. all peaks in a given sample move past the detector region at a velocity equal to the flow velocity of the mobile phase.

assuming an infinitely narrow (d-function) detector. R. representing the various zones formed in the separation process. There are alternative measurements of peak width. In this paper. concentration-sensitive detector. The migration time is the time required for the peak maximum to reach the detector. the migration time of a particular zone.3. and that this is the origin of the migration times observed in CZE’.. which in turn move more rapidly than molecules whose electrophoretic motion is in the direction opposite to the electroosmotic flow. or in the number of the theoretical plates for one component versus another. is given by w. to the spatial width of the sample in the migration channel. The peak height is unaffected by this velocity broadening for a &function. the effect on peak widths has not been fully appreciated. by the zone velocity”. The full width is typically measured at the inflection point in the first derivative of the peak profile. . = (L/t. as the spatial width. with units of time.4. The zone velocity is equal to L/t. The conversion from the temporal width of a zone. wd.)% wd (1) where wd is the spatial width of the detector region. W.177 times the width defined above. We refer to this width. N. For a Gaussian peak shape. with units of time. Fig. corresponds to measuring the full width at 0. the time required for them to pass the detector differs. F. The peaks in the electropherogram. Although it has been noted previously that different zones move at different velocities. the most commonly encountered being the full width at half-height. If the detector response has a more complicated dependence. ZARE molecules whose electrophoretic motion is in the same direction as the electroosmotic flow move more rapidly than neutral molecules. the full width at half-height is 1. for a Gaussian curve. Eqn. This causes the temporal widths for the various zones to be unequal. wt. w’s. In Fig.in zone velocity. which. We refer to this corrected width. An electropherogram obtained in a CZE experiment is a plot of the response of the detector system as a function of time. Because the zones do not travel at the same velocity. COLEMAN.with units of length. to give the actual width of the zone on the column. with units of length.607 of the maximum height. we mean by peak width the full width at the inflection point in the first derivative of the peak unless stated otherwise. The correction involves simply multiplying the temporal width. HUANG. then the observed widths are the convolution of the spatial width with the detector response function. It may range from several hundred microns for absorption detectors to less than 50 pm for conductivity detectors.98 X. 2a shows three Gaussian peaks. as the temporal width. wI. as they might appear in an electropherogram. 1 assumes that the detector response can be represented by a step function of full width. the widths have been corrected for differences. where L is the length of the capillary from the injection point to the detector and t. It is important to realize that these different temporal widths may not imply any difference in the zone volume of the various components. variations in temporal widths may arise solely from differences in zone velocities. it is also necessary to correct the peaks for the finite size of the detector region past which the zones move. Therefore. 2b. Correction for size of detection zone In addition to the correction for zone velocity. may be characterized by a migration time and a width.

0. Effect of correcting measured peak widths for zone velocity and detector width. & F x (b) 0. . In the same figure. The sample is dansylated lysine run at different voltages to produce different migration times. run at a number of different voltages to produce different migration times. 2. 3 shows the temporal peak widths. 40 TIME (arb. 80 units) 8 8 8 I 0. we also present the conversion of those widths to spatial widths according to eqn.2 -7s ..PEAK BROADENING IN CZE 99 (a) 0. In these experiments. 0.4. (spatial) peaks corrected for zone velocity and detector width (in mm). as a function of the migration time. 3.4. the injection volume was held MIGRATION TIME (s) Fig. 1. Solid lines are linear regression lines for the appropriate data set.% 3 Ji 3 z B o. 0 = Experimentally measured (temporal) peaks (ins). of time and (b) as a corresponding Fig. 0 I~J. of dansylated lysine. \ 80 Fig.2 0 0 I\/\ 40 LENGTH (arb. w. Plot ofdetector function of electrolyte units) response for three different peaks (a) as a function length that has moved past the detector.

If the injection profile becomes non-rectangular. diffusional broadening and in the column. the zone length is seen to broadening. with smaller injection widths we find that the zone lengths do depend on the time that the samples are in the column. and ai2. 4 shows calculated zone lengths . contain contributions from an extrinsic intrinsic terms. Examination of Fig. The zone length is equal to twice the square root of the variance. If the observed zone shape is Gaussian. all references to peak widths in the remainder of this paper refer to spatial widths. ZARE constant and detection was accomplished by laser-induced fluorescence. 2(2Dt) “* . of course. Peak broadening caused by diffusion and sample plug length Several authors have assumed that one-dimensional diffusion is the major contributor to peak broadening in CZE 1s2s4 .e. we refer to the spatial width of a peak as the zone length. in particular on the Zength of the injected plug. W.. not &functions but have a finite width. In other cases. i. COLEMAN. N. HUANG. the injection width.j/l2 + 0t7. t. then the value of the denominator increases. Unless otherwise noted.. is given by have been applied for zone velocity where Od2iff is the variance due to diffusion. Thus. We emphasize that the observed peak widths have been corrected for both velocity broadening and detector width before we assess the effect of other factors on the peak width. the overall width is then given by W tot - 2(2Dt.)“’ (3) times this quantity. If the zone becomes trapezoidal. The total variance of a zone. 3 shows that when the temporal widths have been corrected for velocity broadening and the finite width of the detector. where winj is the width of the injected plugi8. and two broadening caused by other interactions Fig. F. 2Dt. If the sample plug that was injected were a true S-function. The detector zone width is approximately 7.. to a Gaussian peak with a variance equal to 2Dt. the expression for the variance changes. as a function of injected length for several The full width at half maximum is 1. + Wi2. where D is the diffusion coefficient.177 Thus. The injected sample plugs are.. For convenience. In many experiments. all peaks in this experiment have very similar spatial widths. R.100 x. represents zone-broadening variance arising from interactions between the analyte and the walls of the column. once corrections and finite detector width. al?. For a rectangular injection profile the variance due to injection is equal to W3j/l2. we have noted zone lengths that are approximately independent of the time that a zone has spent in the column.5 pm.j is the variance due to the injection plug length. the extent to which diffusion affects the zone length depends on the volume of the sample plug that is injected. approaching 24 as the zone profile approaches an isosceles triangle. that plug would spread in time. This simple treatment assumes that other factors. such as heating. do not contribute to the diffusion process and that diffusion is the only broadening mechanism that is present.

In this region the zone length is determined almost exclusively by diffusion. at small injection lengths. Calculations are done using eqn. 1200 s (0) and 2400 s (0).8 cm. (3) A region of curvature in which both diffusion and injection length contribute to the zone length. 5 shows the variation of zone length with injection length for several different substances. Diffusional broadening may be neglected. The qualitative agreement between the experimental data and the model is quite good for injection lengths less than 0. 3. assuming D = 7 lo-’ cm2/s and migration times of 300 s (line). This is followed in each case by a non-linear .)“2. Calculated peak widths as a function of injection width. Fig. The intercept at zero injection ’ I” . Trace (b) is a blow-up of (a) for short injection widths. peak widths may provide a reliable measure of the injection volume.5 INJECTION WIDTH km) Fig.PEAK BROADENING IN CZE 101 0 0. where the zone length is independent of the injection length. + zinc) reduces to 2(20t. These conclusions are strictly valid only if broadening from analyteewall interaction is negligible compared to the broadening from the property being measured. These plots show three distinct regions: (1) A nearly horizontal region at small injection length. By working in region 2. different migration times. In this region and in the limit of no analyteewall interactions the zone length becomes equal to twice the square root of the variance of the injection length. the diffusion width of a b-function injection plug. For each of the substances studied there is a region. 3. 4. By choosing to perform CZE experiments in region 1 it is possible to use the method to determine diffusion coefficients. In the absence of wall interactions this expression width is equal to 2(2Dt. The calculations are based on eqn. (2) A linear region at large injection lengths.

(b) adenosine monophosphate. HUANG. together with estimates of the diffusion coefficients calculated from the Stokes-Einstein equation1 ‘. F. R. Diffusion coefficients are directly proportional to temperature and a portion of the difference between the extrapolated diffusion coefficient and measured . the calculated effective diffusion coefficient is significantly larger than that expected for the systems being studied. Concentrations of each are 1 10m5 M. 3. 2 adequately describes the experimental data. W.2 (cm) Fig. the calculated widths. N. Experiments were run at 20 kV. the extrapolated intercept at zero injection length may be used to calculate an effective diffusion parameter D. The results of these calculations are shown in Table I.4 INJECTION 0. 5. At large injection lengths two of the three systems show a linear dependence on injection length for injection lengths up to 2 cm. The buffer is 20 mM sodium phosphate at pH 7. Assuming that eqn. The zone length as a function of the injection width for (a) adenosine. There are several reasons for this. and (c) pyroximine. COLEMAN. The total length of the capillary is 78 cm and the length from injection to detection is 55 cm. based on the parameters given in Table I and the use of eqn. For two of these species. ZARE 2- (a) 0 0 0.102 X. region. are shown as solid lines. The experimental data are shown by connected circles.8 WIDTH 1.

The temperature change is directly proportional to the power. 4.0056 0.PEAK TABLE BROADENING I AND IN CZE 103 DIFFUSION Substance INTERACTION PARAMETERS Adenosine Adenosine monophosphate Pyroximine 70 16 200 9. 5 that with very large injection plugs. Table I also includes the values of the interaction variance (c&) that give the best lit to eqn. Z2R (where Z = current). as measured . It is also clear from examination of the results in Fig. as the rate of diffusion is greatest immediately following the injection. resulting in a non-Gaussian zone. 5.0036 0. Such a temperature change would produce a maximum increase in the diffusion coefficient of cu. The column turns injection plugs into Gaussian zones through the action of diffusion and what we have called analyteewall interactions. In a typical experiment. largely caused by the temperature dependence of the viscosity. d From eqn.1 0. ’ From Stokes-Einstein equation. this approach is less instructive than that taken above for two reasons.3 7. 3. Sternberg18 has introduced the concept of the column as a Gaussian operator and this is indeed true up to a point. the experimental zone lengths become larger than that predicted by eqn. These values of c& should be regarded as rough approximations because of the uncertainty in the calculated diffusion coefficients. where t. or calculated values presumably lies in this temperature dependence. is the migration time for each species. An effective diffusion coefficient greater than a true diffusion coefficient implies that the analyte-wall interaction term cannot be neglected under conditions where the injection length is a negligible contributor to the zone length.076 a From intercept in Fig. First of all. 5 and eqn. that process will be incomplete. c 2Dcalctm. temperature effects alone cannot account for the differences between the effective diffusion coefficients and the diffusion coefficients of the analytes.011 0. we observe that the temperature in the capillary rises approximately 12 K (from 298 to 310 K) over a period of less than 10 s for an applied voltage of 20 kV (current = 30 PA). where & is used for 2Dt. If the injected plug is too long.0086 0. While avoiding the calculational difficulties discussed above. The actual effect would be somewhat smaller. the measured peak will approach a limit where the width..2 9.036 0. it lumps together several effects in the effective diffusion coefficient. As the plug becomes longer. Therefore. 3. 3 for the species in Fig. and secondly it implies that diffusion and these other effects have the same dependence on migration time. The concept of the effective diffusion coefficient can lead to an alternative expression for the zone length U’ tot = 2(2D’t* + W~“j/l2)“’ where LY is the effective diffusion coefficient. Our treatment has assumed that the zone is Gaussian. 35%.

For smaller plugs. This observation has important implications for the use of CZE in quantitative studies. the concentration (c) profile is given as a function of time by c(x. They have pointed out that this expression is valid if . HUANG. R. it appears that an injection plug greater than 2.t) = &{erflh . is greater than 4. 3. Typical injection lengths are 5-10 times larger. and pL. depending only on the injection width. 3. will approach that of the injection plug. Under controlled conditions. Under these conditions.~/2(Dt)“~] + erf[x + x/2(Dt)“‘]} where x is the position along the column. h. assuming a constant injection volume. undergoes diffusion. The definition of a large injection plug depends on the migration time. For many species. Fig. The zone length is seen to be independent of the concentration. 5 to find the width at the inflection point in the first derivative and by simulating the injected plug as a series of lo-100 b-functions and numerically evaluating the width. Under such conditions peak heights should be directly proportional to concentration. the concentration of the sample should have no effect on the zone length. of a given zone and on the distance from the injection to the detector. by explicit differentiation of eqn. the situation is more complicated because plugs of varying length are seen to give rise to zones of equivalent width. 3 versus injection length. or the diffusion width. a measurement of the zone length allows an estimate of the volume of sample injected. as is demonstrated in Fig. COLEMAN. N. are the electrophoretic and electroosmotic mobilities. the minimum injection length is approximately 200 pm.5 times the diffusion width of a zone is sufficient to qualify as large.104 X. the zone variances should be nearly equal to the variances of the injection plugs. on the order of or less than the diffusion length. In both cases. When a rectangular plug of finite width. 6b. 6a shows the zone length for adenosine as a function of concentration. Number of theoretical plates Jorgenson and Lukacs’ separation. For very small injection lengths the amount actually injected may be dominated by mixing at the interface between the capillary and the sample solution. and V the applied voltage. It also depends on negligible analyte-wall interactions. as have given the number of theoretical plates in a CZE where p. and begins to have a width greater than that given by eqn.. when the injection width. N. W. respectively. ZARE from the electropherogram and corrected as described above. F.5-5 times the intercept of the plot of eqn. and not by the contact time between the sample and the capillary hydrostatic or electrokinetic injection. The measurement of injection length For large injection plugs. Winj in eqn. We have calculated the widths of these concentration profiles (peaks) in two ways. We estimate that for 75pm capillaries. the resultant zone ceases to be Gaussian. co the initial concentration and erf the error function of the appropriate argument 2o. such as those used here.

diffusion is the only source of peak broadening.5 mm. Experiments were run at 20 kV.PEAK BROADENING 10 IN CZE 105 (a) P 5 F !!! Y 2 e . Both of these conclusions have their origin in the fact that the definition of the number of plates is N = L2/02 where o2 is the variance of the peak width broadening mechanism then (zone length). 6. in all cases. they inferred that separation efficiencies are enhanced for substances with small diffusion coefficients and that the number of plates increases linearly with the applied voltage. From eqn. 5- CONCENTRATION ht.4) Fig. The buffer is 20 mA4 sodium phosphate at pH 7. 4. Injection width is the same. If diffusion (7) is the only CT = (20432 where (8) (9) . 6. Effect of adenosine concentration on (a) zone length and(b) peak height. The total length of the capillary is 78 cm and the length from injection to detection is 55 cm.

the zone length is controlled by diffusion and what we have called wall interactions. in the work of Wallingford and Ewing2’ separations in 12.6-pm capillaries. the expression for N still contains the term in a$. . describes the plate number in these experiments. 9. As we have shown. the number of plates is the same for each component of the mixture. the expression for N approaches N = 12L2. N depends on sample concentration and on the total amount of sample only as those quantities are reflected in the injection plug length. there appear to be few.. R. the number of theoretical plates varies linearly with the applied voltage only in the case of very small injection lengths. Even in that instance. F.JV + 1Vi2. 13 is applicable. where eqn. approximately 2. not the plug volume. N.?. 2(2Dt. 7 shows the number of plates for adenosine as a function of applied voltage. 6 or eqn. situations in which the zone length is controlled by diffusion alone! At small injection lengths.j 12 + & 1 (12) In almost all previous experiments. eqn. 13. and length of the capillary from injection to detection. in CZE. Again.5 mm. 12. 6. that is important in determining the zone length.3 times the diffusion width for observed migration time. Thus. In this limit. In such situations. rather than eqn. The essentially complete lack of dependence of N on Vand the quadratic dependence of N on L imply that eqn. we emphasize that it is the plug length. the injection plug length was large relative to diffusion length. In the more common case. and in the absence of additional interactions that influence the zone length. V. It N = (Fe+ PeqLi4 20 (10) Only in the limit that L approaches I do we recover the formula of Jorgenson and Lukacs. Fig.l is the total length of the capillary follows that so that V/l is the electric field strength. ZARE In eqn. while at larger injection lengths the zone length is almost completely determined by the variance of the injected plug. For example.1W&j the on an the (13) Thus. W. an expression for N must involve the total spatial variance of the zone as follows: N = L’/‘(Oiiff + Gf”j+ o..106 X.) ‘I2 . the reported injection volume of 430 pl yields injection length of 3.) (11) from which it follows that N= (pe L2 2DLI + &. L. HUANG. COLEMAN. 22 that N is not proportional careful studies. if any. This to V in their explains the observations by Tsuda et al.

-• . v is the average zone velocity and v1 and v2 are the velocities of the two zones in question 23. In (a) the capillary length is 78 cm overall and 55 cm from injection to detector. .5 mm. R. Using eqn.)v+3T+6:“’ 1 I(v2 Vl)l I(v2 + VI) 2DLl (15) . Dependence of theoretical plate number.+pe. between two zones 1 and 2.PEAK BROADENING IN CZE 107 (a) . and (b) capillary length from injection to detection. 7. L. 4. Resolution Often a more useful index for assessing the performance of a CZE separation the resolution. is used in all cases. Av is the absolute difference in the zone velocities. which is defined as R = l/4 N”’ = l/2 c2 is @v/v) [(I v2 Vll)/(V2 + VI>1 (14) where N is the average number of theoretical plates. The sample is adenosine and the same injection width. on (a) applied voltage. 12 we can express R as R = (L/2) 112 w?nj (p. N. In (b) the applied electric field is 200 V/cm and the length of the capillary between injection and detection is varied. 30 000-_1 APPLIED VOLTAGE (kV) 01 0 500 LENGTH 1000 15 (mm) Fig. The buffer is 20 mM sodium phosphate at pH 7. V.

Dependence of resolution. INJECTION WIDTH ~XI) Fig. R.5 mm and the wall interaction term is set equal to zero.15 10e4 cm’ V-r SK’. The injection width is set at 4. the analyte-wall interaction is only important at small injection lengths. 15. HUANG. 15 using appropriate parameters from Fig. The diffusion coefficient is that of adenosine. 9. as shown in Fig. ZARE 5 10 15 (kV) 20 APPLIED VOLTAGE Fig. R. . It is important to note that voltage influences resolution only in the case of small injection volumes. R.0 and 3. according to eqn. N.108 X. F. 8. 1O-4 cm2 V-’ s.‘) and electrophoretic mobilities of 3. on injection width at two different applied voltages: 10 kV (line) and 20 kV (0). 7a. V. W. Fig. 10 shows the number of plates as a function of the injection plug at 20 kV and an analyte-wall interaction comparable to that observed experimentally for adenosine. on applied voltage. 8. 8. The capillary parameters are those from Fig. Dependence of resolution. Again. This plot is calculated from eqn. This expression leads to the voltage dependence shown in Fig.0. COLEMAN. 9.The calculation is for the resolution between two species that have the same electroosmotic mobility (3.

i. the number of plates is the same for each zone. but quite different in specifics. Inc. As long as the injection length is several times the diffusion length for the species in the zone. Jr. on injection width for no analyte-wall interaction (0) and for analyte-wall interaction (solid line) appropriate for adenosine (see Table I).0 0 0. This work was supported in part by Beckman Instruments. An understanding of the mechanisms for peak broadening in CZE defines the practical tradeoff between improved sensitivity and improved resolution. Applied voltage is assumed to be 20 kV. CONCLUSION We have developed a model for spatial peak widths (zone lengths) in CZE that is similar in nature. WFC acknowledges Wellesley College and NSF grant CHE-85-05926 for sabbatical leave support. quantitation should be easier with larger injection lengths. = ~~inj/31iz.e. Roach and M. as this situation minimizes the effects that arise from differences in electrical properties between the zones and the background electrolyte in the migration channel.. Gordon for many useful discussions. It is expected to become more valid as the concentration of the species in the zones decreases. Pentoney. A. u’~. This model will be useful in designing CZE experiments.2 0. N. 10. particularly in cases where the method is to obtain improved quantitation or to improve resolution. The model was developed neglecting effects of heating and electrical distortions.4 0. Although such operating conditions do not produce the largest number of theoretical plates..8 (cm) INJECTION Fig. ACKNOWLEDGEMENTS We thank S. . J.. M. to the Van Deemter equation. In addition.6 WIDTH 0.. We have shown that the length of the injection plug is often the most significant factor in determining the lengths of the individual zones in CZE under typical experimental conditions. Dependence of theoretical plate number. the observed zone lengths will be approximately equal to the injection length divided by the square root of 3. L.PEAK BROADENING IN CZE 109 0 I I I I J 1.

J. Gozel. Anal. Chromatographia. F. Yagi. A. Jia-Li Liao. Appl. Chromatogr. S. Kuo and R. E. in press. Ando. COLEMAN. Gassmann. E. J. 59 (1987) 44. A. Nakagawa.. Katz. M.-D.C. M. J. Wallingford and A.. M. 2 (1966) 206-270. F. Ewing. F. Chromatogr.C. D. 0. R.. K. Chromafogr. Biochem. Grushka.). Eng. Stokes. Anal. W. 403 (1987) 47. Sierbert and M. Hjertin. 2. and R. 242 (1988) 224. Chromatogr. Oxford University Press. Anal. 60 (1988) 1972. Tsuda. E.. Science (Washington. S. W. Hjertin. Sepaniak and R.). Zuiderweg and A. 1959. J. F. E. Jr. D. W. 270 (1983) 51. Robinson and R. Grant. Chem. W. Kilar. N. Chem. Cole. Jorgenson and K. N. . Sci. Vol. 61 (1989) 241. J. D. Gassmann. Chem. A. 5 (1983) 330. J. Lukacs. C. M. Topics in Bioelectrochemistry and Bioenergetics. A.. M. Zare. 59 (1987) 472. C. New York. X. C. Hjerttn. T. H. K. Anal. 33 (1961) 829. P. 9 (1967) 122.. New York... P. 1975. E. Huang. J. Chromatogr. Milazzo (Editor). J. J. Zare.. R. Sci. Olefirowicz. 222 (1983) 266. Zare. Chem.). Wallingford and T. H. Academic Press. New York. 169 (1979) 11. G. Giddings. Anal. 2nd ed. Chem. The Mathematics of Diffusion. Everaerts and Th. Ewing. D. Anal. Mikkers. Rev.. G. J. Science (Washington. R.110 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 X. H.. Chromatogr. Wallingford and A. J. G. M.C.. Anal. Knox and I. ZARE J. K. Sep.. 89-128. Science (Washington. F. Pentoney. H. Chress. J. J.. Crank. J. 1978.. van Deemter. A. 230 (1985) 813. pp. Kirkland. Otsuka and T. J. Sato and K. 61 (1988) 251. 61 (1989) 292A. Ogan and R. HUANG. Chem.. Zhu. 24 (1987) 135. Gordon. S. Wiley. Michelsen and R. R. Chem. 5 (1956) 271. N. Chem. McCormick and J. Sternberg. L. Elenbring. 4 (1969) 181. E. G. E.. M. R. J. N. S. Jones. C. A. Ewing. P. Scott. J. Verheggen. S. J. P. Terabe. Adv. Adv. Electrolyte Solutions. Klinkenberg. in G. R.

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