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BACKGROUND OF THE INVENTION
The present invention relates in general to a method and apparatus for
controlling liquid flow in capillary electrophoresis (CE).
CE is a known technique for effecting separation of a mixture of analytes
in which a voltage is applied across a capillary containing a sample, and
the resulting electric field causes electrophoretic flow of charged
molecular species in the sample. This technique can be utilized, for
example, to separate proteins having different charges because the applied
electric field will cause the differently charged proteins to travel at
different velocities, thereby causing separation of them along the length
of the capillary.
The charged ions have an electrophoretic mobility which is proportional to
the magnitude of their charge density, and this is one of the major forces
for causing separations in CE. Another force results from the ionization
of silanol groups along the wall of the capillary. In particular, when a
fused silica capillary is employed and is filled with an aqueous solution
having a pH above approximately 2, the surface silanol groups will become
ionized, resulting in a negative charge on the wall of the capillary.
Cations (positively charged ions) of the aqueous solution are attracted to
the negatively charged capillary wall thereby forming an electrical double
layer at the capillary wall solute interface. When a voltage is applied
across the capillary, these cations flow towards a cathode end of the
capillary, thereby resulting in a bulk flow of fluid in this direction.
This bulk flow is referred to as electroendosmosis or electro-osmotic
flow, otherwise known as EOF.
Electrophoretic mobility and EOF are therefore the two major electrical
forces in CE. This can be illustrated by assuming the typical situation
where the EOF is greater than the electrophoretic mobility of the
materials in the sample to be analyzed. Cations are electrophoretically
mobilized toward the cathode end of the capillary, and their
electrophoretic mobility towards the cathode is enhanced by the EOF. On
the other hand, anions (negatively charged ions) are electrophoretically
attracted toward the anode end of the capillary, but since the EOF is
greater than the electrophoretic mobility, the anions net movement is
toward the cathode with the bulk flow. In this situation, the order of
flow of analytes passing a detector positioned at the cathode end of the
capillary will be cations, then neutral species, then anions. It will be
understood that the higher the magnitude of the EOF relative to the
electrophoretic mobility, the more close together the various analytes
will be as they pass the detector. Thus, if the effects of the EOF can be
reduced, the analytes can be more spread out, thereby increasing the
resolution of the separation.
A number of problems are presented in conducting separations with CE.
First, in order to effect a separation of a mixture of analytes, some
method must be employed to load the analyte mixture sample into the
capillary. The most common methods are electrokinetic and hydrodynamic
sample loading. Electrokinetic sample loading, also called electrokinetic
injection or electromigration, utilizes both electrophoretic and
electroosmotic flow to introduce a sample into the capillary. The inlet
end of the capillary and a power supply's anode are placed into a sample
containing vial, and a voltage is applied across the capillary for a
period of time. The strength and duration of the resulting electric field
ideally determine the amount of the sample introduced into the capillary;
however, there is a bias in this method of sample introduction. Briefly,
cations are introduced into the capillary by virtue of both
electrophoretic mobility and EOF. However, uncharged species and anions
are introduced into the capillary by virtue of only EOF since their
electrophoretic mobility is either zero in the case of uncharged species,
or in the direction of the anode in the case of anions. Thus, anions
migrate into the capillary more slowly than uncharged species because of
the electrical attraction of the anions toward the anode. Therefore, the
different electrophoretic mobilities of the analytes arising from their
different charge densities is a source of bias in sample loading by
electrokinetic injection. Another bias occurs because different
electrolyte buffer solutions have different electrophoretic and EOF rates,
resulting in different amounts of sample being injected.
Several approaches to eliminating these sources of bias in electrokinetic
injection have been reported, all of which implicitly aim to decouple
electrophoretic flow from EOF, thereby accomplishing sample introduction
through EOF alone. This decoupling has in all cases been achieved through
approaches involving alterations to the capillary structure itself, such
as by introducing porous glass or a frit, coupling to an additional
capillary or fracturing the capillary. These approaches all require
specialized capillary manufacturing techniques, are rather complex and
labor intensive, and do not address the problems associated with the
changes in the electrolyte buffer solution.
A nonelectrical sample loading method, such as hydrodynamic sample loading,
avoids all of the problems associated with electrokinetic sample loading.
All hydrodynamic sample loading methods involve, by one means or another,
a pressure differential between the inlet and outlet ends of the
capillary. This can be accomplished simply by raising the inlet end of the
capillary above the outlet end, or through the use of either a pressure
pump or syringe pump at the inlet end of the capillary, or a vacuum pump
at the outlet of the capillary. Most known hydrodynamic sample loading
methods are rather bulky and expensive to implement. Further, all of these
methods suffer from potential band broadening with an attendant loss of
resolution if the pressure differential is so large, or the inside
diameter of the capillary is so small, that the injection front is
distorted. For example, in one experiment with an optimized commercial CE
system using pressures as low as 0.497 psi for sample loading, the
experimentally determined injected volume per unit time deviated from a
calculated theoretical value by 6.1%.
Another problem with conventional CE systems is that of controlling the
effects of the EOF. Because the EOF is a source of a zone broadening in
free zone CE, or disturbs focused zones in isoelectric focusing CE, many
investigators have attempted to eliminate EOF entirely. Reducing the EOF
below zero, i.e., reversing the direction of the EOF, or increasing the
EOF may be advantageous in some applications such as micellar
electrokinetic capillary chromatography (MECC) because resolution may be
increased and analysis time reduced.
A number of techniques have been employed to increase, decrease or
eliminate the effects of the EOF. One such technique involves coating the
inner surface of the capillary with a material whose charge is different
from that of the uncoated capillary. Use of an electrically neutral
coating material would eliminate the surface charge that gives rise to
EOF, while use of the material whose charge is more negative than that of
the inner surface of the capillary would increase the EOF. On the other
hand, use of a material whose charge is positive would reverse the
direction of the EOF. However, the use of coatings to change the EOF has
drawbacks. In particular, the coatings degrade with use over time and
suffer from the complexity involved in the coating procedures. Also, these
methods all result in a capillary with a changed, but not adjustable EOF.
Another technique for changing the EOF involves the use of electrolyte
buffer additives which cause Coulombic repulsion between the capillary
surface and the analytes to reduce the EOF. Other additives may be used to
increase or reverse the EOF. Unfortunately, use of such additives presents
the possibility that the additive may adversely affect the material to be
analyzed. Further, the use of these methods once again does not provide
for adjustment of the EOF during CE. Yet another method for changing or
controlling the EOF involves the manipulation of the bulk flow through the
application of electric fields or temperature gradients, although these
methods tend to be overly complex.
A third problem in conventional CE systems is presented by the need to move
the sample material past a detector of some kind after the analytes
therein have been separated. More particularly, once the individual
components in the analyte mixture sample have been separated, they are
caused to flow past a detector, such as a UV absorbance, radioactive decay
or fluorescence detector, so that some attribute of the component can be
sensed thereby. Then the component flows out of the capillary into the
reservoir disposed at the outlet end thereof. Electrophoretic mobility,
EOF and pressure have all been utilized to achieve this mobilization
through the capillary. The same problems associated with sample loading
occur in mobilization; namely, electrophoretic mobilization may be biased,
EOF is difficult to control and pressure systems have been relatively
crude and inaccurate. Once again, none of these approaches allows the
real-time control of the rate of mobilization.
An example of the mobilization problem occurs in isoelectric focusing (IEF)
CE. In IEF CE, a pH gradient is formed along the length of the capillary
and analytes migrate through this gradient until they reach the pH zone
where their net charge is zero, and they stop moving. Thus, the EOF needs
to be eliminated so that there is no bulk fluid flow. In this manner, an
analyte is focused into a zone at its isoelectric point. After the
separation is completed in this manner, the zones must be mobilized past a
detector, ideally without any disturbance of the focused zones.
For this purpose, an electrophoretic mobilization technique called salt
mobilization has been developed and commercialized. Once focusing is
completed, the electric field is turned off, salt is added either to the
anode or cathode buffer reservoirs and the field is reapplied, thus
causing an excess of either H.sup.+ or OH.sup.- to enter the capillary
thereby changing the pH gradient and causing migration toward the cathode
or anode as the case may be. This system is ideally set up so that the pH
gradient will flow past the detector window on its way toward the
electrode. This procedure suffers from the need to perform the multiple
steps discussed above. In addition, the focused zones are susceptible to
diffusion while the field is turned off and to an electrophoretic bias
during the electrically driven mobilization. Hence, reproducibility and
resolution are often adversely affected.
Another technique used for mobilization in IEF CE is to sharply reduce, but
not eliminate, the EOF, such as through the inclusion of methyl cellulose
in the electrolyte buffer. The reduced EOF can then be employed for
mobilization once the separation is completed, however, this technique
requires a compromise between the separation resolution and the
mobilization speed. Further, the rate of mobilization cannot be adjusted
with this technique.
SUMMARY OF THE INVENTION
In view of the foregoing drawbacks to conventional CE systems and
techniques, what is clearly needed is a new CE method and apparatus
whereby sample loading and mobilization can be easily and quickly
accomplished and the effects of the EOF can be increased, decreased or
eliminated as desired during a separation.
In view of the foregoing, it is an object of the present invention to
provide a CE method and apparatus in which a sample to be separated can be
quickly and easily loaded into a capillary, and then mobilized past a
detector once separation of the analytes in the sample is completed.
It is another object of the present invention to provide an improved CE
method and apparatus in which the effects of the EOF can be changed, i.e.,
increased, decreased or eliminated, as desired during a separation.
These and other objects of the present invention are achieved through use
of a system by which pressure and vacuum (negative pressure) are
selectively and controllably applied to a sample capillary so that sample
loading and mobilization can be easily and precisely controlled, and the
effects of the EOF can be easily and precisely increased, reduced or
eliminated, as desired. The system preferably employs a combination
pressure/vacuum pump in conjunction with a plurality of regulators and
valves connected to one end of the capillary. Through selective
application of positive or negative pressure to the capillary, a sample
can be loaded therein, separated and then mobilized past a detector.
In a preferred embodiment of the invention, the pressure/vacuum pump is a
dual-head diaphragm design in which one head supplies vacuum and the other
head supplies pressure. The two heads are each connected through a pair of
regulators, one for precisely controlling very low pressures or vacuums,
and another for regulating higher magnitude pressures or vacuums.
Electronically controlled valves are employed for selectively connecting
the desired regulated pressure or vacuum to a sealed outlet reservoir in
which the outlet end of the capillary tube is disposed.
In one typical operational mode of the system, a relatively high vacuum is
applied to the outlet reservoir to facilitate sample loading into the
capillary from an inlet reservoir. Once the sample is loaded and sample
separation is ready to begin, a precisely controllable pressure is applied
to the outlet reservoir to counteract the effects of the EOF as desired
during sample separation. When the sample is separated, the pressure is
reduced to allow a low mobilization rate of the separated sample past the
detector.
In the above described embodiment of the invention, the applied pressure or
vacuum is controlled by manual adjustment of the pressure and vacuum
regulators and valves. However, in another embodiment of the invention
employing microprocessor control, the pressure and vacuum can be
automatically controlled in response to measured flow in the capillary
tube. To achieve this, a special flow meter is positioned adjacent to a
portion of the capillary tube, and provides flow proportional signals to
the microprocessor. The microprocessor also receives inputs from a data
entry terminal so that it can be programmed to respond to the flow signals
as desired, and generate control signals for actuating the valves and
regulators. The flow meter is specially designed to detect the very low
flow rates present in the very small diameter capillary tube, and in one
preferred embodiment, employs a pulsed light source to periodically
illuminate the flow, and cause a special marker solution therein to
fluoresce. Light detectors are positioned a predetermined distance on
either side of the light source so that the flow rate can be determined by
measuring the time it takes the fluoresced portion of the sample to travel
from the light source to either detector.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and additional objects, features and advantages of the
present invention will become apparent from the following detailed
description of a preferred embodiment thereof, taken in conjunction with
the accompanying drawings in which:
FIG. 1 is a schematic block diagram of a CE system constructed in
accordance with the present invention;
FIG. 2 is an illustration of a control panel for the system of FIG. 1;
FIG. 3 is a schematic block diagram of a modification to the system of FIG.
1 which provides automatic control of the applied pressure and vacuum in
response to the measured flow rate in the capillary; and
FIG. 4 is an illustration of a flow meter for use in the modification of
FIG. 3.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Turning now to a more detailed consideration of a preferred embodiment of
the present invention, FIG. 1 illustrates a CE system 10 including a
capillary tube 12 having an anode inlet end 14 and a cathode outlet end
16. The inlet end 14 of the capillary tube 12 is in communication with an
analyte mixture 18 contained in a first reservoir 20 which is open to the
atmosphere. The outlet end 16 of the tube 12 is in communication with an
analyte mixture 22 contained in a second reservoir 24 which is sealed with
a septum 26.
As is conventional, a high voltage power supply 28 is connected across the
capillary tube 12 through the analyte mixtures 18 and 22 by means of an
anode 30 and a cathode 32 to supply a high voltage which causes
electrophoretic flow of ions in the mixture sample contained in the
capillary tube 12. Since different analytes in the mixture possess
different electrical charges and therefore have different electrophoretic
mobilities, the application of the high voltage will cause movement of the
different analytes at different velocities, thereby effecting a separation
of the analytes along the length of the capillary tube 12. The power
supply 28 includes an on/off switch 33 which allows selective application
of voltage across the capillary tube 12.
Once the analytes in a mixture sample have been separated, the separated
sample components move past a detector 34 toward the outlet end 16 of the
tube 12 and into the second reservoir 24. The detector 34 can be any
suitable detector means for sensing analyte characteristics, such as a UV
absorbance detector, radioactive decay detector or fluorescence detector.
A key feature of the CE system 10 resides in the provision of a
pressure/vacuum supply system 35 including a controllable combination
pressure/vacuum pump 36 for selectively and precisely applying positive
pressure or vacuum (negative pressure) to the second, sealed reservoir 24
to control sample movement through the capillary tube 12. The
pressure/vacuum pump 36, which by way of example can be an ASF Model 7010
ZD/V, includes a pressure head 38 for supplying pressure and a vacuum head
40 for supplying vacuum. The output from the pressure head 38 is split
between a low pressure regulator 42 and a high pressure regulator 44
connected in parallel. The low pressure regulator 42 is set to regulate
pressure over a range of approximately 0.020 to 1.500 PSI, while the high
pressure regulator 44 is set to regulate pressure over a range of
approximately 0.102 to 10.00 PSI. Similarly, the output of the vacuum head
40 is split between a low vacuum regulator 46 and a high vacuum regulator
48 in parallel. Again, the low vacuum regulator 46 is set to regulate
vacuum over the range of approximately -0.020 to -1.500 PSI, while the
high vacuum regulator 48 is set to regulate vacuum over the range of
approximately -0.10 to -10.00 PSI. By way of example, each of the
regulators 42, 44, 46 and 48 can be an Airtrol Model 4104.
The outputs of the regulators 42, 44, 46 and 48 are each connected through
a corresponding pressure transducer 50, 52, 54 and 56 to a respective
electrically controllable valve 58, 60, 62 and 64. Each of the pressure
transducers 50, 52, 54 and 56 generates an electrical output which is
connected to a respective one of a plurality of liquid crystal displays
66. By way of example, the pressure transducers 50 and 54 connected to the
regulators 42 and 46 can each be a Sensym Model SCX 01 DN, while the
transducers 52 and 56 connected to the regulators 44 and 48 can each be a
Sensym Model SCX 05 DN. Each of the valves 58, 60, 62 and 64 can be a
Clippard Model EV-2, while each of the displays 66 can be a Sensym Model
SCX-LCD.
The outputs from the valves 58, 60, 62 and 64 are combined with a tubing
manifold 67 to a single three-way vent valve 68, which in turn is placed
in communication with the interior of the sealed second reservoir 24 by
means of an outlet tube 70. By way of example, the three-way vent valve 68
can be a Clippard Model EV-3. Also connected to the three-way vent valve
68 by means of the tubing manifold 67 is an air vacuum chamber or
reservoir 72. It is employed for maintaining a vacuum or pressure to be
applied to the outlet tube 70 for sample loading. The air reservoir 72
enables the atmospheric pressure in the outlet tube 70 and second
reservoir 24 to be quickly adjusted during sample loading. Another
electronically controlled valve 74 selectively connects the air reservoir
72 to the tubing manifold 67.
Turning now to FIG. 2, a control panel 80 for controlling supply of
pressure or vacuum to the second reservoir 24 is illustrated. The panel 80
includes a plurality of on-off control switches including a system power
switch 82 for controlling power to the various elements of the
pressure/vacuum supply system 35, an air pump switch 84, and a mode
control switch 88 for actuating the air reservoir control valve 74 and
thereby switching between injection and control modes.
A pressure/vacuum switch 90 selects whether the pressure valves 58 and 60
or vacuum valves 62 and 64 will be actuated, while a high/low pressure
switch 92 selects which of the pressure valves 58 or 60 will be actuated,
and a high/low vacuum switch 94 selects which of the vacuum valves 62 or
64 will be actuated. Finally, a vent control switch 96 controls operation
of the three-way vent valve 68 to selectively connect pressure, vacuum or
the atmosphere through the outlet tube 70 to the second reservoir 24.
Also provided on the control panel 80 are four control knobs 98, one for
each of the regulators 42, 44, 46 and 48. Each of the knobs 98 is disposed
along side a corresponding one of the pressure displays 66, and can be
manually turned to adjust the applied pressure or vacuum as desired.
In the operation of the CE system 10, when it is desired to load a sample
to be separated from the first reservoir 20 into the capillary tube 12,
the pressure/vacuum switch 90 is moved to the vacuum position, the
high/low vacuum switch 94 is moved to the low position, and the control
knob 98 for the low vacuum regulator 46 is adjusted until the desired
injection vacuum is obtained as indicated by the corresponding display 66.
At the same time, the mode control switch 88 is moved to the inject
position so that the injection air reservoir 72 will be communicated with
the low vacuum regulator 46 and will be evacuated to the chosen vacuum
magnitude. The vent switch 96 is then moved to the apply position so that
the three-way vent valve 68 places the air reservoir 72 and low vacuum
regulator 46 in communication with the outlet reservoir 24. The air
reservoir 72 is employed to insure that the outlet tube 70 and outlet
reservoir 24 will be quickly evacuated when the three-way valve 68 is
opened. Typically, a sample can be loaded into the capillary tube 12 in
this manner by applying a vacuum of approximately -0.25 PSI for a few
seconds.
Once the sample is positioned in the capillary tube 12, voltage is applied
by the power supply 28 across the capillary tube 12 through the anode 30
and cathode 32 so that separation of the analytes in the sample will
occur. To reduce or counteract the effects of the EOF during the
separation, pressure can be applied to the outlet reservoir 24 as desired.
If it is desired, for example, to counteract the effects of the EOF, a
neutral marker can be injected into the capillary tube 12 ahead of the
sample and caused to move to the detector 34 at which point the pressure
is adjusted so that the neutral marker is held stationary in the detector
window. Since the marker has no charge and is therefore acted upon only by
EOF, the stationary position of the neutral marker is an indication that
the EOF has been counteracted. Once a sample has been separated, the
pressure is reduced in the outlet reservoir 24 so that the separated
analytes flow through the capillary tube 12 past the detector 34.
Experiments have been conducted employing the CE system 10 for different
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