Capillary electrophoresis method and apparatus for electric field uniformity and minimal dispersion of sample fractions

A capillary electrophoresis method and apparatus for reducing dispersion of sample fractions are disclosed. The capillary tube in which the electrophoresis is performed has been flared, at least at its sample entrance end, to remove sharp corners which contribute to aberrations in the electric field distribution in a radial direction and result in differential migration of molecules depending on their proximity to the sharp corners. The flared tube, in contrast to a conventional tube, provides a more uniform electric field for electrophoresis and reduces undesired dispersion of the samples.

DESCRIPTION 
1. Technical Field 
The invention relates to devices and techniques for electrophoretic 
separation of samples. 
2. Background Art 
Applications for electrophoresis, an analytical technique for separating 
biologically important molecules in a sample, include determining a 
sample's homogeneity, determining molecular weights of proteins and 
nucleic acids, mapping nucleic acid primary structures, i.e. DNA and RNA 
sequence analyses, and defining phenotypic variance of protein at the 
molecular level. Electrophoretic techniques rely on the fact that each 
molecular species has a unique combination of mass, size, shape, charge, 
density, and sub-unit structure, with the unique combination creating 
mobility differences responsive to an electric field. Various 
electrophoretic techniques use one or more of these properties to cause 
varying degrees of molecular separation via the migration of the molecular 
species under a constant or varying electric field. 
Capillary electrophoresis is a technique using a capillary tube which is 
filled with a separation matrix, such as a gel or buffer fluid. A small 
amount of sample is introduced into one end of the capillary tube and a 
potential difference is applied along the tube. Differences in the 
electrophoretic mobilities of different molecules cause the fractions of 
the sample to emerge separated at the outlet end of the capillary tube. 
Migration of the various fractions as sharp bands and their emergence over 
a relatively short, clearly defined time is desirable for separation and 
later identification purposes. If, on the contrary, sample constituents 
are widely dispersed in the capillary tube, the constituents are difficult 
to identify and quantify. 
Establishing the proper electric field is one requirement for achieving an 
accurate analysis of a sample. The potential difference applied along the 
capillary tube creates an electric field along the longitudinal axis of 
the tube. For example, a capillary tube having a length of 25 cm will have 
an electric field magnitude of 200 V/cm when a potential difference of 5 
kV is applied along the capillary tube. Varying the longitudinal electric 
field will vary the migration rate of sample constituents, or fractions. 
In addition to the longitudinal electric field, there is also a radially 
extending electric field. A charge accumulation at the interior tube 
surface results from preferential adsorption of anions from the buffer 
solution that fills the migration path of the capillary tube. The negative 
charge of the anions attracts a thin layer of mobile positively charged 
buffer ions. The radially-oriented electric potential is referred to as 
"zeta potential." 
One potential effect of a nonuniform electric field distribution across a 
capillary tube is that individual molecules of a particular sample 
constituent may be accelerated at different rates. Variations in 
acceleration broaden the constituent bands of a sample, thereby decreasing 
the separation efficiency of the process. This may be particularly 
troublesome in the capillary volume context, since samples are often 
obtained in very limited volumes and are often subjected to other 
experimental processes before the electrophoretic separation. 
Electrophoretic dispersion of the sample constituents hinders their 
analysis, and may even lead to loss of the sample. 
U.S. Pat. No. 5,290,587 to Young et al., which is assigned to the assignee 
of the present invention, describes a method of making a capillary tube so 
as to decrease the susceptibility of the tube to electrical 
nonuniformities. A resistive coating is formed along the exterior of the 
capillary tube. The resistivity of the coating is uniform along the length 
of the coating, so that a generally uniform electric field is created by 
the application of a potential difference to the coating. The externally 
applied potential difference is vectorially coupled to the longitudinal 
electric field along the migration path of the tube in a manner to achieve 
a desired zeta potential. The Young et al. method of fabricating the 
capillary tube may be used to increase the accuracy of an electrophoretic 
separation. However, other improvements may further enhance accuracy. 
What is needed is a method and system for enhancing uniformity of electric 
field distribution in capillary electrophoresis. 
SUMMARY OF THE INVENTION 
The invention is a method and apparatus in which a capillary tube is formed 
so as to provide a smoothed contour at the end of the tube, at least with 
respect to the transition from the interior surface of the tube to the end 
of the tube. Material is removed from the entrance end portion of the 
tube, as by chemical etching, so that sharp edges to the interior surface 
are removed. In the preferred embodiment, the removal of material provides 
an entrance end portion in which the inside diameter of the tube increases 
with approach to the end of the tube. 
By modeling the electric field distribution in a capillary tube during a 
capillary electrophoresis separation, it has been discovered that the 
shape of the end portions significantly affects the distribution. When a 
potential difference is applied along a conventional capillary tube, sharp 
corners at the end portions tend to define differences in electric field 
concentrations. Because of the nonuniformity at the end portions, ions 
moving past the ends may undergo different accelerations depending upon 
the positions of the ions relative to the central axis of the capillary 
tube. Consequently, dispersion of a sample during insertion into the tube 
may be increased, adversely affecting the overall separation efficiency of 
the process. "Separation" of samples is the desired result of 
electrophoresis, causing fractionation of a sample of mixed components. 
"Dispersion," however, refers to the scattering of molecules of a 
component, and is usually not a desired consequence of the technique. The 
adverse effects are multiplied for applications in which capillary tubes 
are joined together, since each conventionally formed capillary tube will 
define its own electric field concentrations. 
By smoothing the contour of the entrance end portion of a capillary tube in 
accordance with the present invention, the dispersion of a sample during 
insertion is reduced. In the preferred embodiment, material is also 
removed at the exit end portion in order to smooth the transition from the 
interior to an end surface at the exit. If capillary tubes are to be 
joined together, each tube is treated at both ends in order to provide the 
smooth contour. 
In operation, the entrance and exit end portions are inserted into separate 
buffer reservoirs. Electrodes are used to apply a potential difference 
along the capillary tube in order to initiate electrophoretic separation. 
The removal of the sharp edges at the end portions results in a capillary 
tube that is flared at the end portions of its bore and provides a 
uniformity of the electric field distribution in a radial direction. The 
smooth contour may be achieved by a chemical etching process using 
hydrogen fluoride, but other techniques may be employed. 
An advantage of the invention is that sample dispersion is reduced, i.e. 
the bands of sample fractions separated during the electrophoretic process 
are narrower, facilitating identification and quantification of the 
fractions.

BEST MODE FOR CARRYING OUT THE INVENTION 
With reference to FIG. 1, a conventional electrophoretic system 10 is shown 
as including a capillary tube 12 having an entrance end 14 and an exit end 
16. The capillary tube is of the type known in the art. The capillary tube 
may be a fused silica member having an inside diameter of 50 microns and 
having an outside diameter that is in the range of 140 microns to 500 
microns, but these dimensions are not critical. 
An on-column detector 18 is located along the length of the capillary tube 
12. Ultraviolet absorbance, fluorescence, chemiluminescence, refractive 
index, or conductivity detectors are generally used. The optical coupling 
of the detector to the capillary tube permits detection of movement within 
the capillary tube. 
The entrance end 14 of the capillary tube 12 is inserted into a buffer 
reservoir 22a. At the opposite side of the detector 18, exit 16 of tube 12 
is inserted into a buffer reservoir 22b. The buffer reservoirs 22a-b are 
in fluid communication with the contents of tube 12. Reservoirs 22a-b are 
shown within holders 20a-b, respectively. The two holders 20a-b and 
detector 18 are shown as resting on a table 28. 
A high voltage power supply 34 is connected to the reservoirs 22a-b. A 
first lead line 32 is used to apply a first electrode 33 to reservoir 22a. 
In the same manner, a second lead line 36 applies a second electrode 38 to 
reservoir 22b. Power supply 34 provides a potential difference between the 
ends of tube 12. The migration of the electrophoresed sample will be along 
arrow 30, i.e. from entrance 14 of tube 12 toward exit 16. The fractions 
of the sample separate along the length of tube 12 according to one or 
more of the following: charge, mass, size, shape, density, and sub-unit 
structure. 
FIG. 2 shows an end portion of a conventional capillary tube 12 in 
longitudinal cross-section and also shows the electric field distribution 
at entrance 14. The electric field magnitude is visible as lines 40 and 
region 45a-b. Each line is representative of a constant electric field 
magnitude. A sharp corner, shown at 17a-b, exists at the inside diameter 
of tube 12, because of the way in which tube 12 is prepared for usage. 
Typically, the capillary tube is cut or otherwise segmented from a larger 
length of tubing, producing end surfaces that are generally perpendicular 
to the interior and exterior surfaces of the tube. 
The electric field that results upon application of a potential difference 
along tube 12 is nonuniform in a radial direction, having a region of 
especially high magnitude 45a-b at the corner 17a-b of tube 12. This 
nonuniformity causes differential separation of identical molecules, 
depending upon the proximity of the identical molecules to either the 
sharp corner 17a-b or the central axis 19 of tube 12. For instance, at the 
plane x represented in FIG. 2 by line x-x, the molecules will travel at 
different rates according to their locations in the plane because of the 
extremely variable electric field magnitudes present in a radial 
direction. Plane x crosses many lines of constant electric field 
magnitude, thus signifying nonuniform electric field magnitude along the 
plane. This aberration in the separation process contributes to dispersion 
of the sample fractions. 
FIG. 3 presents a flared capillary tube 11, in accordance with the present 
invention. The interior of tube 11 is treated so as to remove its sharp 
edge at the entrance end or terminus and to smoothly contour its surface. 
Chemical etching with hydrogen fluoride is the preferred method of shaping 
tube 11, but other techniques may be utilized, as will be appreciated by 
persons skilled in the art. The resulting entrance opening 15 of tube 11 
is flared, or trumpet-shaped, preferably with an interior surface at an 
obtuse angle to the end surface, as illustrated at 47a-b of FIG. 3. Tube 
11 is preferably not flared drastically to its exterior edges, as that may 
cause breakage and create new sharp corners. A flare ending in an internal 
diameter in the range of two to three times the original internal diameter 
of the tube is preferred. The shape of tube 11 represents a compromise 
between the mechanical considerations of capillary electrophoresis, 
wherein a uniform internal diameter is preferred, and the electrical 
considerations, wherein sharp edges should be removed. The direction of 
sample migration in tube 11, as with tube 12, is along arrow 30. 
The electric field 48 that is obtained through the use of flared tube 11 is 
more uniform. To illustrate, line y-y of FIG. 3, representing plane y and 
incorporating the diameter of tube 11 at the position of line y-y, does 
not cross many electric field magnitude lines, which indicates the 
electric field distribution is relatively uniform in a radial direction 
along plane y. Therefore, a sample that undergoes electrophoresis in a 
system according to the present invention is less likely to be dispersed 
at the entrance and its fractions are more likely to migrate as sharp, 
recognizable bands. The tube 11 may be incorporated into an 
electrophoretic system as by substituting flared tube 11 for 
sharp-cornered tube 12 in the system 10, presented in FIG. 1. The 
teachings of the present invention are also applicable in a larger format, 
such as electrophoresis in a large-sized tube or lane. 
Tube 11 may be similarly flared at its exit end, opposite entrance 15, so 
that the electric field remains uniform in the exit region, instead of 
being influenced by the sharp corner of the exit end. This is important if 
the sample will be electrophoresed up to the exit region, e.g. if detector 
18 is positioned near the exit region, or if the sample fractions are to 
be collected from the exit end of tube 11 or are to pass through to other 
similar tubes that have been joined to tube 11. In FIG. 4, multiple flared 
tubes 11a-c are shown. Each tube is flared at both its entrance end 15 and 
its exit end 21. Tubes 11a-c are aligned axially, i.e. in an end-to-end 
format. They are then butted together and joined, as with couplers 44. The 
sample to be fractionated is introduced at entrance 15a of tube 11a and 
electrophoresis proceeds toward exit 21c of tube 11c in direction 30, as 
before. In the previous practice, when capillary tubes were 
axially-aligned for electrophoresis, the electrical field was non-uniform 
because of the sharp corners where the tubes were joined. 
The present invention provides an effective method of improving 
electrophoretic separation efficiency providing an advance in the analysis 
of small-volume and sometimes difficult-to-obtain samples. 
While perhaps the invention adapts most easily to use in capillary zone or 
capillary gel electrophoresis, the invention may be used with other 
electrophoretic techniques in which a capillary tube is employed. For 
example, the invention may be used with capillary isoelectric focusing, 
which employs separation of sample constituents by isoelectric point in a 
pH gradient formed over the length of the capillary.