Capillary separation system with electric field assisted post separation mixing

A capillary zone electrophoresis (CZE) system provides for rapid, non-turbulent post-separation diffusional mixing of sample effluent with a fluorogenic-labelling reagent permitting sensitive detection of well-defined sample component zones. A separation capillary extends into a mixing capillary so as to define an annular gap therebetween. The effluent of the separation capillary is mixed with the labelling reagent, which is introduced through the annular gap. A power supply and opposing electrodes establish an electric field which induces electro-osmotic flow of the sample and charge-related differential electrophoretic migration to define component zones. The electric field also causes the separation capillary effluent to diverge as it issues into the mixing capillary so as to facilitate diffusional mixing with the reagent fluid flow without causing significant turbulence. Thus, fluorescence labelling is effective with minimum zone broadening. This system combines the high resolving power of CZE separation with the sensitivity of labelled fluorescence detection to attain an improved system for analyzing biological samples.

BACKGROUND OF THE INVENTION 
The present invention relates to chemical analysis systems and, more 
particularly to an analytical instrument in which sample components are 
separated by differential electrokinetic migration through a narrowbore 
capillary. A major objective of the present invention is to provide for 
post-separation mixing of the sample with another fluid to aid in 
identification and quantification of the separated sample components. An 
illustrative example is the post-separation addition of a fluorogenic 
labelling reagent to separated protein components prior to fluorescence 
detection. 
Chemical analyses of complex organic structures has made noteworthy 
advances in biotechnology possible. Biotechnology has provided techniques 
for manufacturing life-supporting medicines and other products which would 
otherwise be in short supply if natural sources had to be relied upon. In 
addition, entirely new medical products are in development which may 
arrest and cure heretofore untreatable diseases. Biotechnology promises 
new products for agriculture which will feed the world's expanding 
populations and which will enhance the ability of famine-prone countries 
to sustain themselves. 
Chemical analysis of biological samples generally involves the separation 
of the samples into components for identification and quantification. 
Capillary zone electrophoresis (CZE) is one of a class of methods in which 
the different components are moved within a narrowbore capillary at 
respective and different rates so that the components are divided into 
distinct zones. The distinct zones can be investigated within the 
capillary or outside the capillary by allowing the components to emerge 
from the capillary for sequential detection. 
In CZE, a sample is introduced at an input end of a longitudinally 
extending capillary and moved toward an output end. Electrodes of 
different potentials at either of the capillary generate the electrical 
forces which move the sample components toward the output end of the 
capillary. This movement includes two distinct components, one due to 
electro-osmotic flow and the other due to electrophoretic migration. 
Electro-osmotic flow results from charge accumulation at the capillary 
surface due to preferential adsorption of anions from the electrolyte 
solution which fills the capillary bore. The negative charge of the anions 
attracts a thin layer of mobile positively charged electrolyte ions, which 
accumulate adjacent to the inner surface. The longitudinally extending 
electric field applied between the ends of the capillary by the electrodes 
attracts these positive ions so that they are moved toward the negative 
electrode at the output end of the capillary. These positive ions, 
hydrated by water, viscously drag other hydrated molecules not near this 
inner wall, even those with neutral or negative net charge. The result is 
a bulk flow of sample and the containing electrolyte solution toward the 
output end of the capillary. Thus, electro-osmotic flow provides a 
mechanism by which neutral and negatively charged, as well as positively 
charged, molecules can be moved toward a negative electrode. Typically, a 
CZE capillary has a bore diameter of less than 200 .mu.m and preferable 
less than 100 .mu.m, to ensure that the outer molecules interact 
sufficiently with more central molecules to effect an electro-osmotic flow 
which is fairly uniform across the capillary cross section. 
Superimposed on this electro-osmotic flow is the well known motion of 
charged particles in response to an electrical field, commonly referred to 
as electrophoretic migration. The electrolyte solution acts as the medium 
which permits the electric field to extend through the capillary between 
the electrodes. Positively charged molecules migrate toward the negative 
electrode faster than the mean flow due to electro-osmotic flow. 
Negatively charged molecules are repelled by the negative electrode, but 
this repulsion is more than compensated by the electro-osmotic flow. Thus, 
negatively charged sample molecules also advance toward the negative 
electrode, albeit more slowly than the positively charged molecules. 
Neutral molecules move toward the negative electrode at an intermediate 
rate governed by the electro-osmotic flow. 
After a sufficiently long migration through the separation capillary, the 
different sample components separate into bands or zones due to the 
differential movement rates as a function of species-specific charge. An 
appropriately selected and arranged detector can detect these zones 
seriatim as they pass. Components can be identified by the time of 
detection and can be quantified by the corresponding detection peak height 
and/or area. In some cases, the bands can be collected in separate 
containers for a distinct identification and/or quantification process. 
There are several types of detectors used to detect proteins in capillary 
separation systems. Ultraviolet absorbance (UV) detectors are among the 
most common. Other electro-magnetic absorbance detectors could be used. In 
addition, chemi-luminescence, refractive index and conductivity detectors 
have been used. All these methods lack the sensitivity required to detect 
many peaks in CZE protein analysis. High sensitivity is required because 
the quantity of the total sample is limited, and the detector must be 
capable of detecting components that make up only of fraction of the total 
sample. Limitations on sample quantity stem from the requirement that the 
sample be dissolved in electrolyte and that the concentration of the 
sample be low enough to avoid perturbation of the electrical field which 
would lead to distortion of the separated component zones. The sample 
quantity is further limited by the capillary bore diameter and by the 
necessity of confining the sample initially to a relative short 
longitudinal extent. The initial sample extent governs the minimum zone 
breadth and thus the ability of the system to resolve similarly charged 
sample components. 
The detector must be able to detect small quantities of the component in 
each sample zone. A UV detection system is faced with low concentrations 
and very short illumination path lengths and typically yields a poor 
signal-to-noise ratio. Other detection methods are similarly limited. 
Thus, while CZE is effective in separating protein components, there has 
been a limitation in finding a sufficiently sensitive detector for 
identifying and quantifying the separated components. 
Fluorescence detection has been applied in conjunction with liquid 
chromatography (LC), a class of alternative component separation 
techniques. In liquid chromatography, a liquid "mobile" phase ushers 
components through a capillary at different rates related to the 
component's partitioning between the mobile phase and a stationary phase. 
Zones thus form as a function of partitioning ratios. The zones can be 
illuminated and the resulting fluorescence detected. Few proteins can be 
detected with sufficient sensitivity using their intrinsic fluorescence. 
However, labelling reagents can be used to enhance protein fluorescence. A 
major advantage of using fluorescence detection is that the increased 
sensitivity required by small sample quantities can be achieved by using 
very intense illumination. Thus, fluorescence detection used with 
labelling reagents promises to enhance the ability to identify and 
quantify sample components. 
Unfortunately, liquid chromatography is not well suited for high resolution 
separation of proteins. While partitioning ratios differ among components, 
the molecules of any one component at any given time will be divided 
between the mobile phase and stationary phase, and thus move at different 
rates from each other. Despite averaging effects over the length of the 
capillary, sufficient zone broadening is induced by the partitioning to 
prevent high resolution separation of protein components. Since its only 
source of zone broadening is longitudinal diffusion, CZE represents an 
approximately ten-fold improvement in zone-breadth-limited resolution over 
liquid chromatography. 
Fluorescence detection of proteins is not used in conjunction with CZE for 
a number of reasons. As in liquid chromatography, use of the fluorescence 
intrinsic to proteins in not generally applicable. Preseparation 
fluorescence labelling is incompatible with CZE for several reasons. For 
example, pre-separation labelling of protein components causes 
same-species molecules to have different charges. Thus, one component 
separates into multiple peaks, rendering detections virtually 
uninterpretable. Furthermore, sensitivity problems are aggravated because 
each peak represents only a fraction of a sample component. 
Post-separation labelling involves the introduction of fluorogenic 
labelling reagent after separation and before detection. Post separation 
mixing is addressed by Van Vliet et al, "Post-Column Reaction Detection 
for Open-Tubular Liquid Chromatography Using Laser-Induced Fluorescence", 
Journal of Chromatography, Vol. 363, pp. 187-198, 1986. This article 
discloses the use of a Y-connector for introducing reagent into the 
effluent of a separation capillary. One problem with the Y-connector is 
the inevitable turbulence that occurs as the streams merge at an oblique 
angle. The turbulence stirs the sample stream, severely broadening the 
component zones. This broadening can be tolerable in a low resolution 
system, but not in a high-resolution CZE system. 
Post-separation mixing is also addressed by Weber et al. in "Peroxyoxalate 
Chemilumininescence Detection with Capillary Liquid Chromatography" in 
Analytical Chemistry, Vol. 59, pp. 1452-1457, 1987. Weber et al. disclose 
the use of a Teflon tube to convey the separated sample components 
emerging from a liquid chromatography capillary, packed with silica 
particles to the interior of a mixing capillary. An annular gap between 
the Teflon tube and the mixing capillary is used to introduce 
chemi-luminescence reagent coaxially of the sample emerging from the 
narrower (0.2 mm) Teflon tube and into the (0.63 mm) mixing capillary. 
(Note that chemi-luminescence can not be employed in protein component 
detection.) Turbulence is minimized since the reagent flow is fast enough 
to define a sheathing flow confining the sample. However, a problem with 
the sheathing flow is that mixing occurs slowly. Sufficient mixing of the 
chemi-luminescence reagent with sample components thus requires a 
relatively long mixing interval and large mixing volume, during which zone 
broadening in the absence of impairs resolution significantly. While this 
zone broadening may be tolerable in the relatively low resolution liquid 
chromatography system disclosed, it would negate the advantages of a high 
resolution CZE system. 
Thus, one obstacle to post-separation fluorescence labelling in high 
resolution systems is the attainment of rapid, yet low-turbulence and low 
volume, mixing of reagent and sample. However, CZE and other 
electrokinetic separation techniques face another obstacle to 
post-separation introduction of fluorescence labelling reagents, as well 
as other fluids. Fluid introduction generally requires apertures and other 
material inhomogeneities in capillary walls defining the sample path. In a 
CZE separation system, these inhomogeneities can cause field perturbations 
which interfere with electro-osmotic and other electro-kinetic effects. At 
a minimum these perturbations cause zone broadening, but can even 
partially or completely impair electro-kinetic movement of sample 
components. 
In summary, CZE provides a separation technique which affords the 
resolution required for the analysis of complex proteins, but lacks a 
sufficiently sensitive compatible detection technique. Fluorescence 
detection provides a desirable level of sensitivity, but the required 
labelling has not been workable in the CZE context. What is needed is a 
system which combines the resolving power of CZE with the detection 
sensitivity of available with fluorescence-labelled proteins. 
SUMMARY OF THE INVENTION 
Basically, the present invention provides a system which permits 
post-separation introduction of a mixing fluid in a sample stream. The 
geometry and dimensions of the junction permitting this introduction are 
selected so that electro-kinetic effects are minimally impaired. In fact, 
the electric field can be used synergistically to facilitate diffusional 
mixing of sample and mixing fluid, keeping zone broadening to a minimum. 
Thus, the present invention provides for an effective combination of the 
resolving power of CZE separation with the detection sensitivity of 
fluorescence detection. 
Preferably, the effluent end of a electrokinetic separation capillary is 
inserted into a mixing capillary, defining a region of overlap. An annular 
gap between the outer surface of the separation capillary and the inner 
surface of the mixing capillary serves as a port for introducing a 
fluorogenic-labelling reagent or other detection fluid. Electrodes are 
arranged relative to the separation capillary and mixing capillary so that 
an electric field extends from a positive electrode, through the bore of 
the separation capillary, radially across the annular gap, and through the 
bore of the mixing capillary to a negative electrode. The annular gap has 
a sufficiently small radial extent that the electric field is not 
substantially impaired by the gap. Thus, charged molecules of the 
separation capillary effluent are guided along the electric field across 
the annular gap and across the flow of the mixing fluid. Thus, the 
electric field acts to facilitate diffusional mixing of the effluent and 
mixing fluid. Therefore, the mixing is rapid and minimally turbulent, 
enhancing detection without significant zone broadening. 
The Einstein equation for diffusion, x=(2Dt).sup.1/2, establishes practical 
limits on the diameters of the separation and mixing capillaries required 
for sufficiently rapid diffusional mixing. The inner diameter of the 
mixing section of the mixing capillary should not exceed 200 .mu.m and the 
maximum inner diameter of the separation capillary should not exceed 100 
.mu.m so that mixing times are limited to about a second. Preferably, the 
inner diameters are relatively similar. Of course, this requires a 
correspondingly thin wall for the separation capillary in the region of 
overlap. Such a thinned wall can be obtained by chemically etching a 
capillary to the desired extent. 
In the application of primary interest herein, the mixing fluid is a 
fluorogenic-labelling reagent. The present invention allows this 
fluorogenic reagent to be mixed quickly with the sample effluent with 
minimal peak broadening. The small sample volume of a very low diameter 
separation capillary can be compensated by using intense radiation to 
stimulate fluorescence. Thus, the problem of the conflict between 
resolution and sensitivity is largely overcome. These and other features 
and advantages of the present invention are apparent from the description 
below with reference to the following drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
A capillary zone electrophoresis (CZE) system 100 comprises high voltage 
supply 102, a first high voltage electrode 104, a first electrolyte 
reservoir 106 containing a solution of electrolyte 108, a separation 
capillary 110, a mixing tee 112, a mixing capillary 114, a fluorescence 
detector 116, a second electrolyte reservoir 118 also containing 
electrolyte 108, and a grounding electrode 120. Electrolyte 108, which 
also fills most of separation capillary 110 and mixing capillary 114, 
serves as a medium for the electric field between electrodes 104 and 120. 
The same electrolyte is used as a solvent carrier for the biological 
sample to be analyzed. A sample reservoir 122, with a second high voltage 
electrode 124 inserted therein, contains the sample solution 126. A 
reagent reservoir 128 contains reagent 130 which is directed along a 
reagent capillary 132 to mixing tee 112 for mixing with the effluent of 
separation capillary 110 within mixing capillary 114. Reagent flow is 
controlled by a pressure applied to reagent reservoir 128. 
Sample solution 126 can be introduced into separation capillary 110 by 
inserting its input end 134 into sample solution 126. Voltage supply 102 
is activated to establish an electric field from high voltage electrode 
124, through separation and mixing capillaries 110 and 114, to grounding 
electrode 120. As electrolyte is drawn toward grounding electrode 120 by 
electro-osmotic flow, sample solution 126 is drawn into separation 
capillary 110 at its input end 134. Voltage supply 102 is turned off at 
the end of time interval required to introduce the appropriate amount of 
sample solution 126, which can be about 2 nanoliters. 
Input end 134 of separation capillary 110 is then inserted into the first 
electrolyte reservoir 108, to establish the configuration illustrated in 
FIG. 1. With voltage supply 102 again activated, the established electric 
field induces an electro-osmotic flow. Superimposed on this flow are 
relative electrophoretic migration rates which depend on the magnitude and 
sign of molecular charges. The result is that each sample component moves 
at a characteristic rate through separation capillary 110. The 
differential movement rates cause the sample components to exit separation 
capillary 110 into mixing capillary 114 and pass by fluorescence detector 
116 at successive times. 
Fluorescence detector 116 illuminates labelled sample components within 
mixing capillary 114 using a well-focused, high-intensity ultraviolet 
light, such as a mercury xenon arc lamp or a laser. Detector 116 includes 
a photo-multiplier tube which converts the resulting fluorescence 
intensity into a photo-current which is used to obtain an intensity vs. 
time output such as that shown in FIG. 2. The peaks correspond to 
different sample components, e.g., whale skeletal muscle myoglobin (WSM), 
carbonic anhydrase (CAH), .beta.-lactoglobulin B (BLB) and 
.beta.-lactoglobulin A (BLA). Specifically, the conditions were: 0.01% 
(weight/volume) WSM and CAH, 0.005% (weight/volume) BLA and BLB; operating 
and reagent electrolyte buffer 0.05M borate-0.05M KCl pH 9.5; 5 mg 
o-phthaldialdehyde (OPA) +50 .mu.L mercaptoethanol+100 .mu.L ethanol 
diluted to 4 mL with electrolyte buffer; sample introduction 2 s at 30 kV; 
run voltage 30 kV. 
The resolution required to resolve the WSM and CAH peaks and the BLA and 
BLB peaks is obtained in part by using a small bore separation capillary. 
A bore diameter of 100 .mu.m or less permits the electro-osmotic flow to 
act uniformly throughout the capillary cross section and prevents 
convection-induced zone broadening. Diameters smaller than 100 .mu.m can 
be preferred to provide greater electrical resistance between electrodes 
104 and 120. The greater resistance permits greater voltage for a given 
current. It is necessary to limit current to avoid boiling of the 
electrolyte. The higher voltage induces more rapid migration. More rapid 
migration results in less zone broadening due to diffusion (which is 
time-related) without compromising peak separation. 
The small sample volume available using small bore separation capillary 110 
allows relatively little material to be available for detection. In the 
illustrated case, the sample proteins are diluted in electrolyte solution, 
and each peak of FIG. 2 represents only a fraction of the protein content 
of the sample. When UV absorption was used as the detection method for the 
same sample, the component peaks could not be distinguished clearly from 
other peaks due to noise. A similar problem would apply if the 
fluorescence detector had to rely on intrinsic fluorescence of the 
proteins. As indicated above, fluorescence labelling of a sample before 
separation is not a viable alternative. Consequently, the present 
invention provides a novel junction for permitting post-separation 
fluorogenic labelling. This is done in such a way as to minimize zone 
broadening while permitting a sufficiently strong component peak signal 
for identification and quantification of sample components. 
Post-separation labelling is performed using the junction 336 illustrated 
in FIG. 3A. Stainless steel mixing tee 112 has two in-line ports 338 and 
340 and an orthogonal port 342. Separation capillary 110 is supported by a 
first ferrule 344 where it extends through one in-line port 338, while the 
mixing capillary 114 is supported by second ferrule 346, where it extends 
through second in-line port 340. Reagent capillary 132 enters the short 
orthogonal port 342 where it is secured by a third ferrule 348. Fused 
silica reagent capillary 132 has an inner diameter of 200 .mu.m, an outer 
diameter of 325 .mu.m, and a length of 70 cm. Taking the direction of 
sample flow to define a longitudinal direction, then, in accordance with 
the present invention, separation capillary 110 extends into mixing 
capillary 114 so that the two are longitudinally overlapping, defining 
overlap region 350, and preferably concentric. Overlap region 350 includes 
an output section 351 of separation capillary 110 and an input section 353 
of mixing capillary 114. 
In overlap region 350 is defined an intermediate annular gap 352, 
illustrated in FIG. 3B, which provides fluid communication between mixing 
capillary 114 and a mixing section 354, shown in FIG. 3A, within mixing 
capillary 114 near the effluent end 356 of separation capillary 110. This 
permits the fluorogenic reagent 130 to mix with separation capillary 
effluent after sample component separation. After sufficient mixing, 
detection, i.e. sample illumination and fluorescence detection, can occur 
through a detection window 358 downstream of the mixing section 354. 
The preferred embodiment is shown in greater detail in FIG. 4. The 
separation capillary includes a central electrophoretic capillary bore 
460, 25 .mu.m in diameter, a fused silica wall 462, extending radially 
from the 25 .mu.m diameter to 120 .mu.m diameter. Separation capillary 
wall 462 is coated with a protective polyimide plastic coating 464, which 
has been removed near an exposed section 466 of separation capillary 110. 
Within exposed section 466, fused silica wall 462 is tapered to an outer 
diameter of 40 .mu.m, which is the constant diameter of separation 
capillary output section 351. The inner diameter of the mixing capillary 
is then 50 .mu.m. 
Separation capillary 110 was formed by modifying a commercially available 
capillary tube having the dimensions of sepration capillary 110 as shown 
in FIG. 4 where plastic coating 464 is in place. The modification begins 
by stripping the coating over what will become exposed section 466 and 
then etching output end 468 in a stirred bath of concentrated (48%) 
hydrofluoric acid. During etching, water flows through separation 
capillary 110 toward the etchant solution to prevent interior etching. 
Mixing capillary 114 has a bore 470 with inner diameter of 50 .mu.m. A wall 
472 defining bore 470 of the silica mixing capillary 114 has an outer 
diameter of 120 .mu.m. It is important that the walls of separation and 
mixing capillaries 110 and 114 be of similar materials to enhance the 
continuity of the surface charge and thus electro-kinetic effects across 
intermediate annular gap 352; actually, fused silica is used for all three 
capillaries 110, 114 and 132, due to its flexibility, transparency, 
electrical insulation. A polyimide plastic coating 474 extends the outer 
diameter to 150 .mu.m. Detection window 358 can be formed by burning off a 
1-2 cm section of polyimide coating 474. 
Serendipitously, the electric field in overlap region 350 causes sample 
components to diverge radially across the trajectory for the reagent fluid 
flow. The electric field between high voltage electrode 104 and grounding 
electrode 120, shown in FIG. 1, establishes an electrical field 576, FIG. 
5, through separation capillary bore 460 and mixing capillary bore 470, 
effectively defining a path for sample molecules. Electric field diverges 
radially at mixing section 354 and so guides separation capillary effluent 
678 radially outward across the reagent flow 680 and toward the inner 
surface of the mixing capillary, as indicated in FIG. 6. Diverging 
effluent 678 facilitates diffusional mixing without undue turbulence. 
Electric field 576 thus assists diffusional mixing without significantly 
broadening component peaks. Accordingly, system 100 is well-suited for 
high-resolution protein analysis. 
Use of coaxial junction 336 affords mixing of the o-phthaldialdehyde (OPA) 
reagent with migrating sample component zones without excessive zone 
broadening. Detector 116 is linear over three orders of magnitude and 
shows detection limits for amino acids and proteins in the femtogram 
(attomole) range. Other details relating to separation system 100 are 
described in "Instrumentation, Detection and Surface Deactivation in 
Capillary Zone Electrophoresis", by Donald J. Rose, Jr., a Ph.D. 
dissertation submitted to The University of North Carolina at Chapel Hill, 
(March, 1988), which dissertation is incorporated herein by reference. 
Several alternative junction types are provided for by the present 
invention. Two commercially available capillaries can be used with 
complementary dimensions to form the inventive junction, as shown in FIG. 
7A. For example, a separation capillary 782 can have a constant inner 
diameter of 25 .mu.m, a constant outer diameter of 150 .mu.m, while the 
mixing capillary 784 has an inner diameter of 200 .mu.m. In an alternative 
embodiment, the effluent end 786 of a separation capillary 788 is tapered 
to fit within a mixing capillary 790, as shown in FIG. 7B, rather than of 
constant outer diameter. Experimental results indicate that electrical 
field continuity is enhanced and turbulence is further minimized by 
decreasing the difference between the two inner diameters. By removing an 
outer coating from a section of a separation capillary 792, as indicated 
in FIG. 7C, so that its outer diameter is 110 .mu.m in the region of 
overlap, one can use a mixing capillary 794 with a smaller inner diameter, 
for example, 160 .mu.m. 
The preferred embodiment, which is presented again for comparison in FIG. 
7D, provided the most rapid and effective mixing. It is noted that the 
preferred embodiment had the minimum difference between inner diameters 
and thus the minimum average radial distance of effluent dispersion. In 
addition, the reagent and sample flow rates were most closely matched in 
the preferred embodiment. 
The present invention also provides for other mixing section 
configurations. For example, mixing fluid can be introduced in a gap 
between two capillaries of similar inner diameters, the opposing ends of 
the capillaries being adjacent rather than overlapping. Alternatively, a 
single capillary can be used to provide both separation and mixing 
sections by forming an aperture in the wall of the capillary; mixing fluid 
can then be introduced into the sample stream through the aperture. 
Choice of labels is limited by constraints of compatibility with separation 
process. Most fluorogenic labels are themselves fluorescent and thus add 
one or more peaks to detector output. To avoid the spurious fluorescence, 
the reagent must be completely reacted or excess reagent must be removed 
before detection. Both these alternatives are highly problematic. It is 
preferable to use fluorogenic labelling reagents which, like OPA, are not 
themselves fluorescent until they react with primary amine functions of 
protein molecules. 
Generally, the present invention works best when the inner diameter of the 
separation capillary is less than 100 .mu.m and the inner diameter of the 
mixing capillary is less than 200 .mu.m. In addition, the cross-sectional 
area of the annular gap should be between 1 and 4 times that of the 
separation capillary. In the illustrated embodiment, the cross sectional 
area of the separation capillary is about 500 .mu.m.sup.2 and the cross 
sectional area of the intermediate annular gap is about 700 .mu.m.sup.2, 
for a ratio of about 1.4. 
There are altenative electrokinetic separation techniques to CZE. Capillary 
polyacrylamide gel electrophoresis uses electrophoretic migration through 
a gel matrix. Capillary isoelectric focussing distributes sample 
components by isoelectric point in a pH gradient formed over the length of 
a capillary. Isotachophoresis distributes sample components by mobility. 
Micellar electrokinetic capillary chromatography is a form of 
chromatography which uses a "stationary" phase which is subject to 
electro-osmotic flow. All of these separation techniques require an 
electric field to cause movement and separation along capillaries. 
Accordingly, the present invention readily provides for the 
post-separation addition of a detection fluid in conjunction with the 
methods. The present invention can be applied to other capillary 
separation techniques by implementing an electric field to facilitate 
mixing, even though the electric field is not required for separation. 
In the preferred embodiment, a fluorogenic labelling reagent is added after 
separation to enhance detection. The present invention accommodates other 
detection methods and thus the introduction of detection fluids adapted 
for these detection methods. For example, mass spectrometry can be used to 
analyze separated components. The present invention can be used to 
introduce a detection fluid, specifically, a carrier fluid, to sweep 
separated components into a mass spectrometer. These and other variations 
upon and modifications to the described embodiments are provided for by 
the present invention, the scope of which is limited only by the following 
claims.