Method for sample analysis using capillary electrophoresis

A method for performing capillary electrophoresis with improved regularity of migration time of the analytes is disclosed. A capillary electrophoresis system includes a fused silica capillary, a high voltage power supply, electrolyte reservoirs at both ends of the capillary, means for injecting a sample, and a detector. After separation, analytes within the sample are identified by comparing their migration time with the migration time of internal or external standards. Since migration times are dependent on the concentration of the injected sample, identification of unknown analytes can often be difficult or subject to error without the use of such standards. These problems associated with concentration dependent migration time are substantially eliminated by using a combination of high voltage power supply running modes (i.e. constant current, constant voltage or constant power) during the course of the analysis. The control of the high voltage power supply can either be set before a run begins, or controlled directly using feedback from the separation in real time.

BACKGROUND OF THE INVENTION 
Capillary electrophoresis (CF) has become a technique of great interest for 
the separation and analysis of many kinds of ionic species, including 
inorganic ions, biological mixtures such as proteins, peptides and nucleic 
acids, detergents, organic acids and many compounds of pharmaceutical 
nature. Capillary electrophoresis offers the benefits of high resolving 
power, rapid separations, ability to analyze very small volumes of sample 
and a desirable simplicity from the point of view of the apparatus 
required when compared to competing analytical techniques such as liquid 
chromatography. 
The benefits of capillary electrophoresis mentioned above derive to a large 
extent from the use of narrow diameter capillary tubes in the practice of 
capillary electrophoresis. The narrow diameter tubes permit efficient 
removal of the heat generated in the separation process and prevent 
convective mixing which would degrade the separating power. The narrow 
diameter tubes also allow high voltages to be used to generate the 
electric field in the capillary while limiting current flow and hence heat 
generation. The electric field in the capillary tube which provides the 
driving force for the separation is produced by the application of high 
voltage power to the ends of the capillary. The high voltage power is 
generally applied from a high voltage power supply operating at an 
operator selected constant voltage during the separation. 
Variations on the application of constant voltage operation have been 
proposed in the past. For example, in order to overcome drift in certain 
detectors used in CE, Morris et al applied an AC voltage superimposed on a 
constant DC voltage to drive a CE separation, as described in U.S. Pat. 
No. 4,909,919. The AC voltage modulates the velocity of the migrating 
sample components and the detector signal is synchronously demodulated to 
cancel out drift due for example to temperature fluctuations in the 
capillary. Likewise, in applications involving the separation of DNA 
fragments in gel containing capillaries, the high voltage can be applied 
in a pulsed manner as described in U.S. Pat. No. 5,122,248. The pulsing of 
the applied voltage results in a greater degree of resolution of the 
closely related fragments, due to reorientation of analyte molecules in 
the separating medium during different phases of each pulse. However, in 
each of these illustrated examples, the average DC level of the output 
voltage from the power supply remains constant throughout the analysis. 
In other cases, it may be preferable to operate the high voltage power 
supply at a selected level of current through the capillary tube during 
the entire separation. This is referred to as constant current operation. 
Takao Tsuda describes certain advantages of operating at a constant 
current in an article in the Journal of Liquid Chromatography, volume 12 
(1989) page 2501, primarily related to the fact that the separation is 
less dependent on the temperature of the capillary in constant current 
operation. Similarly, constant power operation is also possible where the 
level of power dissipated in the capillary is held constant at a selected 
level. 
The separation that occurs in capillary electrophoresis is generally 
monitored somewhere along the length of the capillary tube by a detector 
that responds with a signal that is proportional to the concentration at 
the monitoring point of the analyte(s) within the sample to be measured. 
Absorbance detectors are commonly used but other kinds of detectors are 
also possible, such as fluorescence, conductivity, electrochemical and the 
like. In some cases, the separating medium is modified prior to detection 
by the addition of reagents to label the analyte or by modifying the 
conductivity of the medium through ion exchange. 
The detectors employed are to a greater or lesser extent non-specific. That 
is, they respond with an indication of the concentration of many different 
analytes thus not directly determining the identity of the analyte. In 
some cases, additional information is available to aid in identifying the 
sample components, such as when the detector is capable of measuring 
absorbance at multiple wavelengths so as to obtain an absorbance spectrum 
or ratios of absorbances at several wavelengths. Alternatively, the 
separated analytes can be collected as they emerge from the capillary and 
subjected to further analysis by other techniques. However, 
multiwavelength detection adds cost and complexity and spectral 
information may not be available if the analyte is transparent over the 
spectral range being measured and is being determined instead by an 
indirect absorbance measurement where the response is due to the 
displacement of an absorbing species in the separating medium. Post 
separation analysis by other techniques requires additional time and 
effort and is not always possible due to the small amount of sample 
analyzed by capillary electrophoresis. 
The detector also permits the measurement of the migration time for each 
analyte and the migration time can be used to characterize each component 
of the sample mixture. For purposes herein, the migration time for each 
analyte is defined as the time period from the start of the analysis to 
the appearance at the detector of the concentration peak of that analyte. 
The migration time of each sample component can be compared to the 
migration times of known standards, and when combined with other knowledge 
about the origin and character of the sample, allow the identity of the 
sample components to be inferred. In order for the migration time to be 
useful, each analyte must appear in the separation at its characteristic 
migration time over the range of experimental conditions that the analysis 
is to be used for. In particular, the migration time of each analyte must 
remain essentially constant over the range of sample compositions that are 
likely to be encountered. 
It is known that the migration time of each analyte tends to fluctuate 
depending on the composition of the sample. Petr Gebauer, Wolfgang 
Thormann and Petr Bocek provide a theoretical explanation of how such 
fluctuations can occur in an article in the Journal of Chromatography, 
volume 608 (1992) pp. 47-57. This article describes how the concentration 
of a major component in the sample can affect the migration time of minor 
sample components by amounts in the tens of percent and cautions that the 
record of each analysis must be evaluated with great care to avoid making 
a false determination of the identity of the analytes in the sample. 
However, the authors do not propose solutions to prevent migration times 
from varying. Also, neither the patent cited above concerning the 
application of pulsed voltage nor that describing the imposition of an AC 
voltage on the DC separation voltage addressess the problem of migration 
time variation caused by changing sample composition. 
In certain applications, the effect of sample composition changes can be 
magnified, as when it is desired to maximize the sensitivity of a 
capillary electrophoresis analysis. For example, it may be advantageous 
for the sample mixture to be analyzed to be dissolved in pure water or 
other very low conductivity medium rather than, for example, in the 
separating medium. This causes a phenomenon known as stacking to occur and 
permits a larger volume of sample mixture to be introduced into the 
capillary without undesirable broadening of the analyte concentration 
peaks. However, when the sample is dissolved in pure water to achieve this 
desirable enhancement in sensitivity, the migration time of the analytes 
becomes even more dependent on the sample composition. 
It would thus be desirable to be able to use migration time as an 
identifying characteristic for sample components in CE and it would also 
be desirable to do this while also using the sensitivity enhancement that 
results from having the sample mixture dissolved in pure water or other 
low conductivity medium. 
SUMMARY OF THE INVENTION 
This and other shortcomings of capillary electrophoresis systems of the 
prior art are addressed by the present invention which includes a 
capillary electrophoresis system incorporating novel methods of control of 
the high voltage power supply. The control of the high voltage power 
supply is characterized by initiating a change in its output condition 
during the analysis, such as the sequential use of two or more of the 
following high voltage operating modes in a single analysis: constant 
current, constant voltage, and constant power. 
The change in output condition is appropriately timed to effectuate the 
substantially complete elimination of migration time changes that are due 
to varying concentrations of ions in the sample. The timing and control of 
the switching of the power supply output condition may be determined by 
calculation prior to the analysis and/or initiated by feedback control 
from the capillary during the analysis. 
In one embodiment, the first time segment of the analysis is conducted in 
the constant current mode, followed by the remainder of the analysis being 
conducted in the constant voltage mode. The length of the time segments is 
determined prior to analysis. Alternatively, the first segment of the 
analysis is conducted in the constant voltage mode, followed by the 
remainder of the analysis being conducted in the constant current mode. 
Again, the segment length is determined prior to analysis. In a further 
refinement of the embodiment, on-line migration time measurements of an 
internal standard are used to determine optimum switching times of the 
high voltage power supply thereby eliminating residual migration time 
shifts of the analytes to be measured.

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION 
FIG. 1 shows a block diagram of a CE system 10 of the present invention. 
The system includes a fused silica capillary 12 with an inside diameter 
having a range of from about 5 to about 500 .mu.m, and preferably from 25 
to 100 .mu.m, having an inlet end 22 and an outlet end 32. Each of the 
ends of the capillary tube is immersed in respective inlet and outlet 
reservoirs 23, 33 containing an electrolyte solution 14 which extends to 
fill the inside volume of the capillary. Contacting the electrolyte 
solution in the reservoirs are separate electrodes 16, 18 that are 
connected to the output terminals of a high voltage power supply 20. The 
high voltage power supply output is set by a controller 30. 
To perform an analysis, a sample solution contained in vial 34 is 
introduced into the capillary 12 by placing the inlet end 22 of the 
capillary into the sample solution in vial 34 after which a hydrostatic 
pressure in the range from 0.1 to 1 psig is applied to the sample solution 
with respect to the outlet end 32 of the capillary. Alternatively, the 
high voltage power supply 20 can be activated for a period of time after 
the inlet end 22 of the capillary has been immersed in the sample solution 
in vial 34, with the resulting electric field causing sample to migrate 
into the capillary. 
After a sufficient amount of sample is introduced, the inlet end 22 of the 
capillary 12 is returned to the electrolyte reservoir 23. High voltage is 
then applied by the power supply 20 to the electrodes 16, 18 contacting 
the electrolyte solution 14 so that the voltage is applied from one end of 
the capillary tube to the other. The controller 30 can signal the power 
supply to apply the voltage in different modes. The term "mode(s)" is used 
to denote the power supply output regulation and is intended to encompass 
constant voltage, constant current and constant power operation. Further, 
the controller can cause the power supply to change mode or output level 
during the analysis. 
The high voltage creates an electric field within the capillary 12 that 
causes the components of the sample to move along the length of the 
capillary tube. The velocity of motion of each component is a function of 
its molecular size and shape, the electrical charge that each molecule 
carries, the viscosity of the electrolyte solution in the capillary tube, 
the magnitude of the electric field in the capillary and the rate of bulk 
flow caused by electroosmosis. A UV/Vis absorbance detector 40 is 
positioned at any convenient location along the length of the capillary, 
preferably near the outlet end 32 and is adapted to measure the components 
of the sample in known fashion through the walls of the capillary. As 
individual sample components pass through the detector segment of the 
capillary, the detector output signal changes in a manner that is related 
to the concentration of analyte molecules passing through that segment of 
the capillary tube. The output signal from the detector is connected to a 
data collection device 50. 
We have studied the effect of sample composition on the migration time of 
the components of the sample and found that the effect of sample 
composition can be correlated to the mode of operation of the high voltage 
power supply. This is illustrated in FIGS. 2a-d and FIGS. 3a-d. A series 
of separations of the inorganic anions, chloride, sulfate and fluoride, 
were performed using an electrolyte of known composition and conductivity 
where the concentrations of chloride and sulfate in the sample solution 
were held constant but the concentration of fluoride was varied. 
Four samples (designated Sample A, B, C and D respectively on the two sets 
of graphs) were composed. The composition of each analyte of interest, the 
numbered peak corresponding to each separated analyte shown on the graphs 
as well as the measured conductivity for each sample and of the 
electrolyte is given below. 
______________________________________ 
Ion Peak Conductivity 
Composition Number (.mu.S @ 24.degree. C.) 
______________________________________ 
Sample A 
Chloride 4 ppm 1 56 
Sulfate 4 ppm 2 
Fluoride 4 ppm 3a 
Sample B 
Chloride 4 ppm 1 203 
Sulfate 4 ppm 2 
Fluoride 30 ppm 3b 
Sample C 
Chloride 4 ppm 1 356 
Sulfate 4 ppm 2 
Fluoride 60 ppm 3c 
Sample D 
Chloride 4 ppm 1 1519 
Sulfate 4 ppm 2 
Fluoride 300 ppm 3d 
Electrolyte 
-- -- 1131 
______________________________________ 
The CE system 10 used for these separations was a Quanta.TM. 4000 equipped 
with a Model 860 data system, commercially available from the Waters 
Chromatography Division of Millipore Corporation. The system was used with 
a Waters AccuSep.TM. fused silica capillary 12 which had a 75 .mu.m inner 
diameter and was 60 cm long. The system was operated under the following 
conditions to obtain the separations shown in FIGS. 2a-d: 
Electrolyte: 5 mM sodium chromate, 0.5 mM tetradecyltrimethylammonium 
bromide (TTAB), pH 8.0 
Sample Introduction: 9.8 cm hydrostatic head for 30 seconds 
Power Supply: negative polarity 
Potential: 20 kV 
Detector: UV Absorbance located at 52 cm from inlet end 
Wavelength: 254 nm 
Detector Time Constant: 0.1 second 
Data Acquisition: 20 pts/sec. 
As indicated, FIGS. 2a-d show the results obtained when the separations 
were performed according to prior art techniques where the high voltage 
power was applied in the constant voltage mode. One sees that the 
migration time of both chloride and sulfate decreases as the concentration 
of fluoride is increased. In comparing FIGS. 2a and 2b we see that the 
migration time of sulfate in FIG. 2b approximates the migration time of 
chloride in FIG. 2a. This is undesirable since it makes identification of 
the chloride and sulfate concentration peaks difficult, particularly when 
there are additional components in the sample. 
The separations shown in FIGS. 3a-d were run under the same conditions as 
above except the power supply 20 was operated in constant current mode at 
20 .mu.A. Here the effect of increasing the fluoride concentration is to 
cause the migration time of both chloride and sulfate to increase. Though 
opposite in direction, this effect is equally undesirable. 
We have found that by properly using more than one power supply operating 
mode in combination during a single analysis, the effects illustrated 
above can be made to cancel resulting in essentially no change in 
migration time for the analytes of interest as the sample composition 
varies. For example, if the system 10 is operated in constant current mode 
for a period of time, the optimum length of which can be experimentally 
determined, and then switched to constant voltage operation for the 
remainder of the separation, the resulting migration times become 
substantially independent of sample composition. 
As previously mentioned, using the prior art methodology, the migration 
times of the components of the sample are dependent on the composition of 
the sample. For the example above, in constant voltage mode, the migration 
times become shorter as the overall concentration of ions in the sample 
increases (and hence the conductivity of the sample increases), whereas in 
constant current mode the reverse happens, namely the migration times 
become longer as the overall concentration of ions in the sample 
increases. 
These observations can be understood on the basis of the magnitude of the 
electric field in the capillary 12 and in the variation of that field 
along the length of the capillary. The electric field, acting on the 
charge that each ion carries, creates the driving force that propel the 
ions in electrophoresis. Any changes in the electric field or its 
distribution will therefore be reflected in changes in the velocity of 
migration of the ions in the sample. 
The following example will show how the sample composition influences the 
migration time of the components of the sample. The same four samples 
(A,B,C and D) corresponding to the samples shown in FIGS. 2a-d and FIGS. 
3a-d, all containing the anions chloride, sulfate and fluoride (each as 
the sodium salt) with the concentration of fluoride varying as shown in 
Table I, were analyzed by the CE system 10. 
TABLE I 
______________________________________ 
Sample and Electrolyte Composition 
Sample [Cl] [SO4] [F] Conductivity 
______________________________________ 
A 4 ppm 4 ppm 4 ppm 55 (.mu.S/cm) 
B 4 4 30 203 
C 4 4 60 356 
D 4 4 300 1519 
electrolyte: 
5 mM sodium chromate, 
1131 (.mu.S/cm) 
0.5 mM TTAB, pH8 
______________________________________ 
Sample introduction into the capillary 12 is by 9.8 cm of hydrostatic head 
applied for 30 seconds. The capillary is 60 cm long with an inner diameter 
of 75 .mu.m. 
The length of the sample zone (i.e. the length of the plug of sample 
solution that flows into the capillary during the 30 seconds that 9.8 cm 
of hydrostatic head is applied) can be calculated. The average velocity, 
v.sub.avg, of the flow is given by the following equation: 
EQU v.sub.avg =.DELTA.p d.sup.2 /32.eta.L 
where .DELTA.p is the pressure difference between the two ends of the 
capillary (.DELTA.p=9.61.times.10.sup.3 g/cm sec.sup.2 at 9.8 cm head), d 
is the capillary inner diameter (75 .mu.=7.5.times.10.sup.-3 cm), .eta. is 
the viscosity of the solution in the capillary (10.sup.-2 g/cm sec for 
water at 20.degree. C.), and L is the capillary length (60 cm). 
Substituting these values: 
EQU v.sub.avg =2.82.times.10.sup.-2 cm/sec. 
The sample zone length, L.sub.s, is given by the product of the velocity 
and the duration of the sample introduction period. 
EQU L.sub.s =(2.82.times.10.sup.-2)(30)=0.842 cm. 
The 0.842 cm length of the sample zone is big by CE standards, representing 
1.4% of the 60 cm capillary length. It is possible in this case without 
undesirable broadening of the sample peaks because the samples are 
dissolved in pure water. This means that the analyte bands, which move at 
velocities different from that of the sample zone, will sharpen as they 
migrate into the electrolyte. 
The electrical resistance of a capillary filled with electrolyte is 
calculated and the effect on the resistance of introducing the sample zone 
for the different sample compositions is determined. The measured value of 
the electrolyte conductivity, .sigma., is 1131 .mu.S/cm. R.sub.cap, the 
resistance of the capillary, is determined from the following equation 
(with terms and values as above): 
##EQU1## 
Similarly one can calculate the resistance, R.sub.s, of the 0.842 cm sample 
zone for each sample as well as the resistance of the remainder of the 
capillary, R.sub.el, and the total resistance of the two zones (i.e. 
sample and electrolyte) in series, R.sub.tot. 
TABLE II 
______________________________________ 
Resistances of the Zones in the Capillary (ohms). 
Sample R.sub.s R.sub.el R.sub.tot 
______________________________________ 
A 3.47 .times. 10.sup.8 
1.18 .times. 10.sup.9 
1.53 .times. 10.sup.9 
B 9.39 .times. 10.sup.7 
1.18 .times. 10.sup.9 
1.27 .times. 10.sup.9 
C 5.37 .times. 10.sup.7 
1.18 .times. 10.sup.9 
1.23 .times. 10.sup.9 
D 1.26 .times. 10.sup.7 
1.18 .times. 10.sup.9 
1.195 .times. 10.sup.9 
______________________________________ 
Since the two zones are electrically in series, R.sub.tot is given by the 
sum of R.sub.s and R.sub.el. R.sub.el is less than R.sub.cap because the 
length of electrolyte is shorter by the length of the sample zone. 
The sample zone with low fluoride concentrations have higher electrical 
resistance. It can be seen that the dilute samples have a significant 
effect on the overall capillary resistance. This means, for example, that 
in constant voltage operation, the capillary current will be significantly 
lower when dilute sample is introduced. 
The electric field strength in the sample zone and in the rest of the 
capillary for the different samples and for both constant voltage and 
constant current operation can be calculated from the equation below: 
EQU E=I R/L 
where E is the electric field in volts/cm, I is the capillary current 
(given by V/R.sub.tot in the constant voltage case, V being the voltage 
applied to the capillary), R is the resistance and L is the length of the 
zone. 
The electric field values are shown in Table III. It is important to 
realize that a low conductivity sample zone never disappears even after 
the analyte ions have migrated out of the zone and been replaced by 
background electrolyte ions, but continues to traverse the length of the 
capillary. While the edges of the zone will blur due to diffusion, the 
sample solvent zone will maintain its identity and move through the 
capillary at the electroosmotic flow rate. Thus the impact of the sample 
zone on the field distribution in the capillary will be felt throughout 
the separation (if, as here, the sample zone elutes after the analyte 
ions) though the resistance of the zone does change somewhat as 
electrolyte ions and counterions replace analyte ions and counterions due 
to differences in the mobilities of these ions. Thus the current trace in 
constant voltage operation shows some rapid changes as the analytes 
migrate out of the sample zone and are replaced by background electrolyte 
and a more gradual change due to the diffusional blurring of the sample 
zone edges and a final value after the analytes have eluted but with the 
sample zone still in the capillary which is different for each of the 
sample levels. 
TABLE III 
______________________________________ 
Electric Field Distribution in the Capillary (V/cm). 
Const. Voltage Const. Current 
Sample E.sub.s 
E.sub.el E.sub.s 
E.sub.el 
______________________________________ 
A 5390 260 6882 333 
B 1756 313 1862 333 
C 1037 323 1065 333 
D 250 335 250 333 
______________________________________ 
For constant current operation, Table III shows that as the sample becomes 
more conductive in going from sample A to sample D, the field in the 
sample zone, E.sub.s, decreases. In the rest of the capillary, because the 
current is always held at the same level, the field, E.sub.el, does not 
change. The overall effect is for migration times to get slightly longer. 
This is what is experimentally observed. 
In the constant voltage case, the situation is a bit more complicated 
because of the way the applied voltage divides between the zones. As in 
the constant current case, the field in the sample zone decreases in going 
from sample A to sample D. But now, the field in the rest of the capillary 
increases as the field in the sample zone decreases. These changes have 
opposing effects on the migration time. Because the analytes traverse the 
sample zone quickly and spend the majority of the migration time in the 
electrolyte zone, the overall effect is for migration times to get 
shorter. Again, this is borne out experimentally. 
There are several points to be made from these results. One is that in 
constant current operation, all the variability is in the sample zone 
field and hence in how fast the ions traverse this zone. This effect is 
illustrated by considering how long it takes chloride with a mobility of 
7.9.times.10.sup.-4 cm.sup.2 /V sec to traverse the 0.842 cm sample zone. 
The results, determined by the equation below, are summarized in Table IV. 
##EQU2## 
TABLE IV 
______________________________________ 
Time (seconds) for Chloride to Traverse Sample Zone 
Const. Current 
Sample E.sub.s 
time 
______________________________________ 
A 6882 .15 
B 1862 .57 
C 1065 1.00 
D 250 4.26 
______________________________________ 
As the fluoride concentration increases, it takes longer for the chloride 
ions to traverse the sample zone and hence the overall migration time 
becomes longer as well. 
Another point is that the total resistance of the capillary changes with 
changes in sample conductivity as we have seen in Table II. This means 
that by measuring the current in constant voltage mode or voltage in 
constant current mode, one can measure the total resistance and from the 
resistance calculate the actual conductivity of the sample (this is done 
by reversing the calculations above leading to the total resistance). The 
value of the sample conductivity may be used in determining how to change 
the separation conditions during the analysis run to achieve migration 
times that are independent of sample composition. In constant voltage 
mode, for example, where low conductivity samples have slower migration 
times, one could increase the voltage partway into the separation with the 
amount of the increase depending on the measured sample conductivity. Such 
correction could also be applied on the basis of direct measurements of 
sample conductivity prior to the analysis. The value of the sample 
conductivity would enable the controller to determine from a lookup table 
stored in its memory the correct time point in the analysis run to effect 
the increase in separation voltage and the amount of increase to be 
applied. 
Since the effect of changing sample composition on the migration time is in 
opposite directions for constant voltage and constant current operation, 
we have found that a particularly advantageous approach to achieving 
migration times that are independent of sample composition is to combine 
these modes. That is, by operating in one mode for part of the analysis 
time followed by a change to another mode for the remainder of the 
analysis time the opposing effects can result in a cancellation of the 
migration time variations. We have seen that in constant current mode, 
migration times increase as the sample becomes more concentrated (and 
hence more conductive) due to the decreasing field strength in the sample 
zone. We have also seen that in constant voltage mode, the field in the 
rest of the capillary increases as the sample becomes more concentrated 
leading to shorter migration times. By starting the analysis in constant 
current mode, followed by a change to constant voltage mode partway into 
the analysis time, the resulting migration times can be held substantially 
constant for the range of sample compositions provided the time of 
switching the mode is properly selected. 
The application of this method to the analysis by the CE system 10 of FIG. 
1 of the same series of samples as in FIGS. 2a-d and FIGS. 3a-d is shown 
in FIGS. 4a-d (with compositions, numbered peaks and conductivity as given 
previously). Here the initial segment of the analysis, 72 seconds in 
duration, is performed in the constant current mode at 20 .mu.A. Then the 
controller changes the high voltage power supply operating mode so that 
the second segment of the analysis, lasting for the remainder of the 
analysis time, is performed in the constant voltage mode at 20 kV. The 
other conditions are the same as in FIGS. 2a-d. As can be seen from the 
results, the migration times for chloride and sulfate remain substantially 
unchanged as the concentration of fluoride in the sample is varied. As a 
result, the concentration peaks can now be easily identified. 
To illustrate the predetermination of the optimum time for switching the 
high voltage power supply operating mode, reference is made to the graph 
of FIG. 5. This graph plots the difference in migration time (.DELTA.MT) 
for both chloride and sulfate ions. Each point on the graph represents a 
.DELTA.MT measurement between sample D of FIG. 2d and FIG. 3d having a 
conductivity of 1519 .mu.S/cm (i.e. a high concentration of fluoride) and 
sample A of FIG. 2a and FIG. 3a having a conductivity of 56 .mu.S/cm (i.e. 
a low concentration of fluoride) as a function of the duration of time the 
CE system was run in the constant current mode at 20 .mu.A, which was 
chosen as the first time segment of the analysis. The second time segment 
was performed in the constant voltage mode at 20 kV. The total analysis 
time was 180 seconds, and all the other conditions are the same as in 
FIGS. 2a-d. As shown at the extreme left (0 seconds constant current 
operation) and right (180 seconds constant current operation) of the 
abscissa of the graph, representing full duration constant voltage and 
constant current operation repectively, there are large differences in 
migration time for the ions between the two samples. Thus, for example, 
when run at full duration constant voltage, .DELTA.MT for sulfate is about 
-4.8 seconds and .DELTA.MT is about -4.3 seconds for chloride. The 
.DELTA.MT's for these ions under full duration constant current operation 
are likewise large but now have a positive shift. Between these extremes 
are the points in time suitable for switched mode operation. At these 
points there is a gradual transition from the negative difference values 
of constant voltage operation to the positive difference values of 
constant current operation. By selecting a value of 72 seconds for the 
duration of the first time segment, the migration time differences are 
near zero for both chloride and sulfate ions. 
Variations on this approach can be used to achieve the same result. For 
example, using the two modes (constant current and constant voltage) in 
the reverse order can also effect the desired result though the time of 
switching modes will be different. Also, one can divide the analysis time 
into more than two time segments and switch back and forth between the two 
modes more frequently. Constant power operation by itself is intermediate 
in its effect on migration time when compared to constant voltage and 
constant current operation. It can likewise be combined with one of the 
other two modes to eliminate migration time differences. 
Further enhancement of the method involves the use of on-line migration 
time measurement and appropriate feedback control to determine optimum 
high voltage power supply switching times. This can be done by 
incorporating a reference peak into the separation such as by adding a 
minor component to the separation medium filling the capillary and the 
electrolyte reservoirs in FIG. 1. If indirect absorbance detection is 
being used, the minor component should be substantially non-absorbing at 
the wavelength being used for detection. Conversely, if direct absorbance 
detection is used, the minor component should be absorbing at the 
measuring wavelength. The incorporation of the minor component will result 
in the appearance of a concentration peak at the detector at a migration 
time characteristic of the minor component. Alternatively, a known 
component could be added to the sample solution. If the minor component 
added to the separation medium or the known component added to the sample 
is chosen to produce a peak before most or all of the analyte peaks, then 
an on-line measure of the migration time of the reference peak can be 
directly measured and used to apply further correction. That is, the 
method detailed above can be used to determine migration times that are 
substantially independent of variations in sample conductivity. Any 
residual migration time change in the reference peak can be directly 
measured and used to apply a correction such as by initiating a further 
change of the high voltage power supply mode. This use of on-line feedback 
control provides second order correction which further eliminates 
migration time changes for the remaining sample components. For example, 
bromide is a rapidly migrating species in the analysis of anions by the 
method illustrated in FIGS. 2a-d. If bromide were added to the sample 
solution, a peak due to the bromide would be expected to be observed in 
the detector output at a given time point before the peaks for chloride 
and sulfate. If after use of the method of switching modes during the 
analysis run outlined above, a residual migration time shift for the known 
bromide concentration peak was detected, the measured time shift could be 
used to initiate another switching of mode to eliminate the residual 
migration time shift for the remaining peaks. That is if the bromide peak 
were detected at a point in time later than expected the power supply mode 
would be switched by the controller 30 back to constant current operation. 
The present invention has been described in terms of constant voltage, 
current or power conditions. The term "constant" is intended to include 
substantially unvarying DC levels as well as any time varying waveform in 
which the average value of voltage or current measured over a complete 
cycle of the waveform remains substantially constant. Examples of such 
time varying waveforms, which are considered to be within the scope of the 
present invention, are the application of a pulsed voltage or current 
waveform to the capillary or the superimposition of an AC voltage or 
current on a DC separation voltage or current. Therefore, it is possible 
to use pulsed or AC modulated waveforms to obtain the same results in 
combining modes to eliminate migration time variations in the ways that we 
have previously described using substantially unvarying DC levels as long 
as the average value of these time varying waveforms, measured over a full 
cycle of the waveform, remains substantially constant for the duration of 
the time segment that mode is applied. 
Although we have illustrated the method of the invention on the analysis of 
selected inorganic anions, it is apparent that the method will have 
applicability to other ions that can be analyzed by capillary 
electrophoresis. Furthermore, other variations of the invention will 
suggest themselves to those with ordinary skill in the art.