 |
Description  |
|
|
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to an apparatus for performing capillary
electrophoresis and more specifically to the optical path for such an
apparatus.
2. Description of the Prior Art
Capillary electrophoresis (CE) is a chemistry separation technique which
utilizes the differences in solute electrophoretic velocity to isolate the
various components of a sample. FIG. 1 depicts a typical CE apparatus. A
high intensity electrical field supplied by high voltage power supply 10
is applied across a teflon, glass, or quartz separation capillary tube 12
of narrow inside diameter (5 to 400 micrometers) containing an
electrolytic buffer solution. For an uncoated, open capillary tube, the
presence of the electrical field imparts motion to charged and uncharged
moieties present in the buffer through two mechanisms: electro-osmotic
(endoosmotic) flow and electrophoretic force. Flow of buffer (or sample
from sample vial 14) through capillary 12 is detected by a detector 16.
Electro-osmotic flow is the bulk flow of buffer from a first buffer vial 18
to a second buffer vial 19 through capillary 12 due to the shearing
movement of a diffuse layer of cations past a more firmly held, dense
layer, interacting with integral, anionic groups of the capillary wall.
Factors which influence the velocity of electroosmotic flow are:
electrical field strength; buffer dielectric constant; zeta potential (the
electrical potential existing between diffuse and compact cationic
layers); and buffer viscosity (which is dependent on bulk properties of
the buffer and the temperature of the buffer). For electro-osmotically
driven, packed capillary, reverse phase chromatography applications,
solvents of use are any normally used solvent for standard reverse phase
liquid chromatography.
Electrophoretic force is the force applied to charged particles residing in
an electrical field, and neutral or uncharged molecules are not affected.
Positively charged molecules (cations) migrate towards the cathode while
negatively charged molecules (anions) move towards the anode. Factors
controlling solute electrophoretic velocity are: molecular charge;
electrical field strength; viscosity of the migration media; and solute
molecular geometric factors.
The net velocity at which a solute travels in an uncoated, open capillary
tube during CE is the vector sum of the electro-osmotic and
electrophoretic velocities. Buffer viscosity plays a significant role for
both of these phenomenon. Both electrophoretic and electro-osmotic
velocities are inversely proportional to buffer viscosity, thus affecting
the net migration velocity for all solutes.
When an electrical field is applied to a capillary which contains buffer,
joule heating occurs. Accordingly the temperature of the buffer within the
capillary increases until a steady state of heat exchange between the
capillary and its surrounding environment is achieved. Consequently the
ultimate buffer temperature is dependent upon the ambient temperature
surrounding the capillary. Because of the temperature dependence of
viscosity, the mobility of a solute in a given buffer within a given
capillary in a given electrical field is largely determined by ambient
temperature. For temperatures between 15.degree. to 30.degree. C., a
1.degree. C. temperature increase results in an approximate 2 percent
decrease in viscosity, increasing solute net velocity by 2 percent.
As is the case in many chromatographic techniques, solute identity is
linked to migration time and velocity. For one form of CE known as
capillary zone electrophoresis, samples are loaded into the capillary as a
slug or plug. The latter may be achieved by application of an electrical
field or some hydrodynamic force (vacuum or pressure head). An electrical
field is then applied and the solutes migrate, as bands, down the
capillary at their respective net velocities. Differences among these
velocities create the primary mechanism for solute separation. These
solute bands are then detected by monitoring a bulk property of the buffer
such as refractive index, photometric absorbance, fluorescence, electrical
conductivity, or thermal conductivity. The time period extending from the
initiation of the separatory process to the point of solute detection is
termed the migration time. The net velocity is determined using the
migration time and the distance traveled by the solute.
Because of the high efficiencies achieved in capillary electrophoresis, it
is not uncommon to see peak widths as narrow as two to three seconds. For
complex solute matrices, multiple peaks may be separated by as little as
two to three seconds in migration time. Consequently, a twenty minute CE
run in which the temperature has changed by 0.1.degree. C. can experience
changes in migration time by as much as 2.4 seconds, possibly causing
improper solute identification. Thus, efficient temperature regulation is
required.
In the prior art, a capillary tube 12 as used in an electrophoresis
instrument is supported in a variety of ways, depending on whether tube 12
is to be cooled by air, by liquid, or by metal plates in contact with the
capillary tube. Cooling of tube 12 is important since the electrophoresis
process subjects the capillary tube to a very high voltage which causes
joule heating in the capillary tube. It is important to maintain the
temperature of the tube at a stable predetermined temperature so as to be
able to make measurements at a known temperature. Various schemes have
been suggested for supporting and cooling the capillary tube, all of which
have significant disadvantages and many of which are not suitable for air
cooling purposes.
Prior art electrophoresis and similar spectrographic instruments typically
include an optical path as shown in FIG. 2, which includes two light
sources 22, 24 each of which provides a different spectra. Typically one
light source 22 is a deuterium (D.sub.2) source and the second light
source 24 is a tungsten (W) light source. A movable shutter 26 is provided
in front of light sources 22, 24 so as to switch in light source 22 or
light source 24 depending on which spectra is desired. A light beam 28
from either light source is then passed through baffles 29 onto a concave
holographic grating 30 or similar diffraction device, and then is focused
into beam splitter 32 through baffles 33.
Beam splitter 32 in one form in the prior art is a short length of optical
fibers. In the typical prior art instrument, a portion of the light
transmitted to some of the optical fibers emerges from the beam splitter
32 at reference arm 34 and is sent via window 36 to a reference
photodetector 38 which detects the reference light beam for purposes of
comparison. The remainder of the light transmitted through beam splitter
32 is transmitted through a longer length of optical fibers to sample end
40 of the beam splitter and is focused using a lens 42 into sample cell 44
in which the sample is held. The portion of the light which passes through
sample cell 44 and the sample therein is then directed onto a second
(sample) photodetector 46 through window 48. The first and second
photodetectors 38, 46 are matched substrate photodetectors, i.e. cut from
the same piece of crystal or other photodetecting material, so as to have
matching thermal properties. Also shown is monochromator casing 50. The
dual beam approach compensates for fluctuations and the changes in
intensity of the light source level, as well as any changes in intensity
in the propagation of light.
For the purpose of remote detection in which only the sample arm is
elongated, this prior art system has several disadvantages. Since
reference photodetector 38 and sample photodetector 46 would be widely
separated, they are subject to different amounts of heat due to their
different locations. Thereby the problem of dark current i.e., drift
caused by unequal heating, is significant, resulting in less precise
measurements. Also, if the sample arm 40 of beam splitter 32 (i.e., that
portion of the optical path which leads to the sample) is mechanically
flexed, this flexing distorts the optical path through the optical fibers
in sample arm 40, resulting in more or less light reaching sample cell 44.
Since the portion of the light beam which reaches reference detector 38 is
not so distorted, this causes a difference between the reference light
beam and the sample light beam. Thereby, the prior art system is deficient
because the common path of propagation is not maintained to the sample 46
and reference photodetectors 38.
Another significant problem with prior art electrophoresis instruments is
the relative difficulty of controlling the temperature of the sample
inside the capillary tube. As discussed above, capillary tubes are
typically cooled by forced air or circulating liquid or by placing the
capillary tube between metal radiator plates. The object is to cool and/or
heat the capillary to a particular target temperature. Typically, the
temperature control of the capillary tube in the prior art is performed by
monitoring the temperature of the media surrounding the capillary tube.
This process is problematic in that a thermal dam occurs at the interface
between the media surrounding the capillary tube and the capillary tube
itself. That is, thermal transfer is inhibited across this boundary, and
therefore the temperature of the media surrounding the capillary tube is
not exactly the same as that of the capillary tube itself.
As discussed above, electro-osmotic flow is the bulk flow of a solution to
the capillary tube under high voltage which occurs in most forms of
capillary electrophoresis in which the interior wall of the capillary tube
has not been treated. It is well known that solutes move through the
capillary tubing under the influence of the applied electric field at a
net velocity equal to the vector sum of the electrophoretic velocity and
the electro-osmotic velocity. Thus a cation, neglecting any solute-wall
interactions, will have two mobilities or velocities in the same direction
and thus will tend to move through the tubing relatively quickly. An anion
will have an electrophoretic velocity which is the vector opposite
direction of the electroosmotic velocity and thus will tend to move
through the capillary tubing relatively slowly. A non-charged species
i.e., a neutral species, will have no electrophoretic velocity at all and
thus can be used to measure the electroosmotic velocity of the system.
Typically amides or some other neutral species are used to measure
electro-osmotic velocity. These materials are typically known as neutral
markers. The term neutral marker refers to the fact that in the buffer of
interest, the neutral marker solute has no electrical charge.
In the prior art, electro-osmotic flow is determined by introduction of a
neutral marker and then observing at one particular wavelength the flow of
the neutral marker through the system to identify when the neutral marker
passes the detector. This process works well with very simple sample
combinations, where no other solutes co-migrate with the neutral marker.
If however other compounds present in the sample combination are also
neutral, this complicates the process of detecting the neutral marker.
It is also known to detect electro-osmotic flow without the use of a
neutral marker. In one known process, the electro-osmotic flow is
determined by the level of current stabilization when different buffer
solutions having different specific conductivities were provided in the
anode and buffer reservoirs. This process relies on the assumption that
the system demonstrates a zeta potential and dielectric constant which is
not seriously affected by the change in the electrolyte composition in the
solutes. In another method, electro-osmotic flow is determined without the
use of a neutral marker by observing continuously the weight of the
material held in the cathode buffer reservoir. The volume transfer is then
determined by dividing the change in mass of the cathode buffer reservoir
by the density of the buffer. These last two methods are extremely time
consuming and difficult and require significant manual intervention in
addition to being of doubtful accuracy. Thus, there is a significant need
for a method to determine the electro-osmotic flow by an automated process
which can deal with complex sample combinations.
SUMMARY OF THE INVENTION
In accordance with the invention, various improvements are made to an
electrophoresis instrument for purposes of improving the accuracy and
usability of the instrument and to allow measurements not obtainable using
the prior art instruments.
In accordance with the invention, capillary tubing is coiled and enclosed
in an air cooled cartridge. The air cooled cartridge includes a housing,
electrodes fitted to the capillary tubing, and a spherical lens assembly
which is part of the optical path. The air cooled cartridge holds the
capillary tubing so as to optimize air cooling of the capillary tubing
when the cartridge is installed in the instrument. The air cooled
cartridge fits into a manifold which includes both an anode and a cathode
subassembly for holding vials containing the sample or buffer solutions
and a ground potential chamber.
Also provided in accordance with the invention is a method of marking the
air cooled cartridges using a bar code so as to provide identifying
information for automated handling of the cartridges.
Also in accordance with the invention, a remote optical path is provided in
which a fiber optic bundle having a particular arrangement of optical
fibers for carrying the sample and reference light beams has an extended
reference arm for carrying the reference light beam to the reference
detector, which is located in close proximity to the sample photodetector.
Thus the reference photodetector is in the same environment, i.e., heat
level, as is the sample detector. This structure is advantageous in that
remote transference of detection light in the common arm of the bifurcated
optical fiber bundle reduces the optical system sensitivity to mechanical
perturbations to the optical fibers. Thus slight changes are viewed
simultaneously by both the sample and reference photodetectors and are
thus more easily corrected.
In accordance with another aspect of the invention, the temperature of the
capillary tubing during electrophoresis is controlled by observation of
the electrical resistance of the capillary tubing. This method relies on
the determination that the electrical resistance of the tubing containing
a given buffer is a unique function of its temperature. Thus resistance
may be calculated from the observed voltage and current across the
capillary, and the capillary tubing may be air cooled by provision of an
air flow across the capillary tubing in response to the observed
resistance.
The provision in accordance with the invention of very precise reproducible
temperature control provides the ability to perform thermal gradient
electrophoresis in the instrument. It has not been possible previously to
perform this process in a reliable, reproducible manner since the required
temperature control equipment was not in existence.
Also in accordance with the invention, a method is provided of determining
electro-osmotic flow by use of a neutral marker in which the spectral
characteristics of the neutral marker are identified and used to determine
when the neutral marker has passed the detector. The method of observation
and determination of the spectrum associated with the neutral marker
allows use of determination of electro-osmotic flow even in the case of
co-elution or co-migration of a solute which is similar in its
electrophoretical profile to that of the neutral marker.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a prior art electrophoresis apparatus.
FIG. 2 shows a prior art optical path for the UV-visible detector which may
be used in the device of FIG. 1.
FIGS. 3(a) to 3(c) show views of an electrophoresis instrument in
accordance with the invention.
FIG. 3(d) shows schematically a column conditioning and hydrodynamic
injection system in accordance with the invention.
FIG. 4 shows an air cooled cartridge in accordance with the invention.
FIG. 5 shows the air cooled cartridge partially inserted into the
temperature control system.
FIGS. 6 and 7 show a temperature control system in accordance with the
invention.
FIG. 8 shows a manifold in accordance with the invention.
FIGS. 9(a) to 9(f) show a remote optical path in accordance with the
invention.
FIG. 10 shows detail of the fiber optic bundle used in the remote optical
path.
FIG. 11 is a flow chart showing a temperature control method in accordance
with the invention.
FIG. 12 shows a calibration plot for temperature control.
FIG. 13 shows a gradient micellular electrophoresis apparatus in accordance
with the invention.
FIG. 14 shows detail of the gradient micellular electrophoresis apparatus.
FIG. 15 is a flow chart showing use of a neutral marker in accordance with
the invention.
Similar reference numbers in various figures denote similar or identical
structures.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the invention, various improvements are provided over
the prior art electrophoresis apparatus.
FIG. 3(a) shows a front view of an electrophoresis instrument in accordance
with the invention. Shown in enclosure 56 is the front panel 58 in a
closed position, air cooled cartridge loading port 60, information
displays 62, 64, and control buttons 66.
FIG. 3(b) shows a rear view of the instrument of FIG. 3(a). Shown in
enclosure 56 are high voltage power supply 70, high voltage line 72, power
transformer 74, vacuum exhaust port 76, oven purge port 78, and helium
inlet port 80. The rear of the detector 82 portion of the instrument is
shown. Also included are RS232 connector 86, I/O port 88, power switch 90,
and voltage selector and fuse block 92.
FIG. 3(c) shows a front view of the instrument of FIG. 3(a), with the front
panel removed. Shown are electric power board 98, a conventional
autosampler 100, buffer solution bottle 102, helium valve 104, dessicant
bottle 106, waste trap 108, injection vacuum tank 110, pressure transducer
112, vacuum pump 114, fluid pump 116, valve 118, beam splitter 120,
optical bench 122, manifold 124, oven (thermal chamber) 126, fan 128, and
air cooled cartridge 130.
FIG. 3(d) shows schematically a column conditioning and hydrodynamic
injection system for the above described instrument in accordance with the
invention. Shown are fluid pump 116, vacuum pump 114, valve VA, valve VB,
valve VD, cathode reservoir 136, vent line 138, valve VE, valve VF, valve
VC, atmospheric pressure line 140, injection vacuum chamber 110, pressure
transducer 112, and control microprocessor 142.
Air Cooled Cartridge
An air cooled cartridge 130 (see FIG. 4) is used for capillary
electrophoresis in accordance with the invention. Cartridge 130 consists
of: a main body 146; a bobbin assembly 148; a spherical lens holder
assembly 150; metallic electrodes 152, 154; electrical contacts 156, 158;
and capillary tubing 162 of glass, quartz, or teflon, typically no greater
than 500 microns in inside diameter and about 10 to 200 cm. long.
Main body 146 is a support for the other subassemblies. Additionally, main
body 146 aligns the electrode 152, 154 and optical 150 subassemblies with
their respective counterparts in the manifold and remote optical path (not
shown here but described below).
Bobbin assembly 148 supports capillary 162 which is coiled in a concentric
circle. Bobbin 148 consists of a central support ring 166 with radiating
capillary support pieces 168, 170, etc. Each capillary support piece 168,
170, etc., contains four equally spaced holes (not shown) and one hole
centered above the array of four, through which capillary 162 is threaded
and held in place. The thickness of each support piece 168, 170 is
minimized, maximizing the capillary surface area exposed to ambient air.
Spherical lens holder 150 fastens capillary 162 to the cartridge main body
130 prior to entry of capillary 162 to electrode 152 as well as holding
capillary 162 in the proper orientation with spherical lens holder 150,
permitting precise image focusing into the capillary lumen, thus limiting
stray light. Spherical lens holder 150 mates with the remainder of the
remote optical path (not shown) to provide precise, reproducible optical
alignment, as described below.
Metallic electrodes 152, 154 are constructed of high conductivity, low
electrochemical reactivity metals. An alloy of platinum-iridium is used in
one embodiment. A portion of capillary 162 exits cartridge 130 and enters
the manifold (not shown) by passing through the center of electrodes 152,
154. Electrodes 152, 154 each have an inside diameter slightly larger than
that of the outside diameter of capillary 162. This minimizes the dead
volume between electrodes 152, 154 and capillary 162.
Air cooled cartridge 130 is a structure approximately five inches (12 cm.)
wide, nine inches (22 cm.) high, and 0.25 inch (0.5 cm.) thick. Main body
146 of the air cooled cartridge is preferably molded from black delrin.
Other low thermal mass, low thermal conductivity, and high dielectric
strength materials may be used. The dimensions of air cooled cartridge 130
may be otherwise as convenient. Air cooling slots 171-1, 171-2, ..., 171-n
are formed in main body 146. Spherical lens holder 150 is preferably made
of black UV stabilized ABS and is a flange-like structure with a smaller
portion which fits inside a cavity provided in main body 146 and with a
lip for fitting against main body 146 to fix lens holder 150 in place.
Other high dielectric strength, UV stable materials may also be used for
lens holder 150.
Bobbin assembly 148, around which capillary tube 162 is concentrically
wound, is formed of delrin and is about 3.5 inches (9.0 cm.) in diameter
and fits inside a cavity provided in main body 146.
As shown in FIG. 5, air cooled cartridge 130 is partially lowered into a
chamber 174 at one side of which is provided a Peltier type heat sink
device 176, which is a well known type of solid state device for cooling
and/or heating to precise temperatures. On each side of cartridge 130 is
an insulative layer of polyethylene 178, 180, each layer 178, 180
approximately 0.78 inches (2.0 cm.) thick. Air cooled cartridge 130 when
fully lowered into position between insulative layers 178, 180 is locked
in place by a cartridge lock bar 182. A retaining thumb screw 184 is also
provided. Also provided is a fan (the blades of which are hidden and not
shown) mounted on panel 187 driven by a regulated DC motor 188 fitted with
a heat sink assembly 186 for drawing the air cooled by Peltier heat sink
176 across the capillary (not shown) in cartridge 130.
The cooling system in accordance with the invention is shown schematically
in FIG. 6 showing DC motor 188 for driving fan blades 189, and also
installed on panel 187 supporting fan blades 189 is a temperature sensing,
resistive thermal device 192 (RTD). As shown, fan blades 189 draw the air
(shown by lines) through the center of bobbin 148. The air is then
recirculated by fan blades 189 across capillary 162 to the temperature
regulating heat exchange surface 197 of the Peltier device.
Peltier device 176 is sandwiched between conventional heat exchanging
surface 197 and conventional heat dissipating/collecting surface 198.
Surfaces 197, 198 are separated by a 0.25" (6.3 mm) thick layer of
polyethylene insulation 199.
As shown in FIG. 7 in a block diagram, RTD device 192 provides a
measurement of the temperature of air surrounding capillary 162. The
capillary electrical resistance is determined by dividing the applied
voltage (usually about 5 KV) by the measured current during a calibration
phase. This resistance information is provided to microprocessor 142 which
is part of the electrophoresis instrument and which in one embodiment is a
Motorola 68008 microprocessor. This microprocessor then uses the ambient
air temperature and capillary resistance data to control the drive current
200 to Peltier device 176 so as to maintain a constant electrical
resistance and set point temperature 202 in capillary 162 during the
separation process. The actual set point is the capillary resistance.
Ambient temperature (not shown) is used as a secondary parameter to
anticipate the arrival at the desired capillary resistance, thus
minimizing set point setting time.
The lower portion of the air cooled cartridge when in a lowered position is
in contact with a manifold 124, as shown in FIG. 8. The air cooled
cartridge (not shown) fits into alignment slots 210, 212. Manifold 124
includes a high potential (anode or cathode) subassembly, including high
voltage contact 214, which accepts vials containing either sample solution
or buffer solution and also includes a central support subassembly 220,
and a ground potential chamber 224, containing high voltage contact 216,
which is connected to a valve assembly via port 226 which allows (under
automatic control) the filling and flushing with buffer and application of
vacuum to the capillary tubing in the air cooled cartridge for the purpose
of rinsing, washing, or hydrodynamic injection. These processes are
performed by the structure shown schematically in FIG. 3(d). Also provided
in manifold 124 is a hole 230 for the optical bench (not shown, described
below) to slide into so as to contact the spherical lens assembly (not
shown, in the air cooled cartridge). A vial-like chamber 232 is built into
the manifold structure so as to eliminate the need for a ground potential
buffer vial. Also provided are high voltage line entrance 236 and high
potential vial holder 238.
Bar Code on Cartridge
In accordance with the invention as shown in FIG. 4, air cooled cartridge
130 is marked with a bar code index 246 at a convenient location to
identify the particular cassette. Also, additional information is included
in bar code index 246. This information includes the length of capillary
tube 162 in that particular air cooled cassette. The length of the tube is
required as described above for determining electrical field strength, and
electro-osmotic and electrophoretic mobilities and velocities. The length
is also required to calculate the fluid-flow resistance in the capillary
tube. The fluid-flow resistance is necessary for the system to determine
automatically how long it takes for the capillary tube to be flushed with
a given solution and what would be the approximate volume of sample loaded
into the capillary tube for a given vacuum applied for a given period.
The system is automatically informed by reading bar code index 246 of the
inside diameter of capillary tube 162. This is necessary for determining
the fluid resistance of the capillary tube and the electrical resistance
of the capillary tube for the above stated reasons.
The system is also informed automatically by bar code index 246 whether the
tube is an open capillary tube, i.e. contains no gel, or is a closed tube,
i.e. contains gel. This is important for hydrodynamic or vacuum type
injections because the gel would be damaged or destroyed by application of
a vacuum or hydrodynamic forces. Bar code index 246 also indicates whether
the tube has its interior lined with a coating for purposes of knowing
whether there is significant electro-osmotic flow in the capillary tube.
Also, as described above, each particular cartridge 130 is identified with
its own particular number in bar code index 246 so that the system can
automatically track the performance of each cartridge and/or capillary
tube based on the separation efficiency for a given test.
Bar code index 246 on air cooled cartridge 130 is read in one embodiment of
the invention by a conventional bar code reader (not shown) which is part
of the electrophoresis instrument. Thus the bar code reader in the
instrument reads bar code index 246 on a particular air cooled cartridge
and provides the information in the index to the microprocessor and
related computer software for the above described purposes.
Remote Optical Path
The remote optical path as shown schematically in FIG. 9(a) includes a
unique fiber optic beam splitter 120 (as in FIG. 3(c)) arrangement for
detecting small sample volumes (down to about 100 picoliters) in a
capillary. Light 260 is focused into a fiber optic bundle 262 from the
exit slit of a conventional monochromator (including one or two light
sources, a shutter, and a diffraction grating as in FIG. 1 and not shown
here). The monochromator may be generating light of a given band-width for
the purpose of UV-visible photometric absorbance detection, fluorescence
detection, refractive index detection, as well as any other means of
photometric detection. Light may also be focused into fiber optic bundle
262 from a coherent light source (laser) for the purpose of refractive
index, fluorescence, thermal-optical-density detection, as well as other
means of coherent light photometric detection. Fiber optic bundle 262 is
remotely bifurcated at point 264 into a sample arm 266 and a reference arm
268. The light exiting from reference arm 268 impinges onto a reference
photodetection device 272 located in the same environment as the sample
photodetection device 274. Light emitting from sample arm 266 is focused
using a plano-convex lens 276 into a second, spherical lens 280 in direct
contact with the capillary 162.
The fibers of fiber optic sample arm 266 may be arranged in cross section
in a circle, rectangle, square, trapezoid, or other parallelogram or
triangular pattern in order to facilitate the focusing of the image into
the center of the capillary. Sample photodetector 274 is placed directly
behind capillary 162. The beam splitter housing self-aligns via locating
hole 230 in the manifold 124 (see FIG. 8) and on the cartridge lens holder
150 (see FIG. 4). Spherical lens 280 is thus located in the cartridge lens
holder 150, while the plano-convex lens 276, reference photodetector 272,
and both ends of the fiber optic bundle 262 are housed in a retractable
member (not shown) which slides into and out of the spherical lens holder
150 which is mounted on cartridge 130 (see FIG. 4).
This structure is advantageous in that remote transference of detection
light in the common arm of a bifurcating fiber optic bundle greatly
reduces the optical system's sensitivity to mechanical perturbations to
the fiber optics. In this approach, light changes are simultaneously
viewed by sample 274 and reference photodetectors 272 and are thus
correctable.
Placement of both photodetectors 272, 274 in a similar environment reduces
perturbations resulting from physicomechanical variances in detection
environments. The combination of lenses produces an image of appropriate
size for small volume detection without any attendant loss of throughput.
The mechanical layout of the system is such so that all optical elements
are self-aligning.
FIG. 9(b) shows detail of the optical path at its upper end down to the
bifurcation point. Shown are beam splitter body 284, an insert 286 in body
284 to hold the optical fiber bundle 262, the optical fiber exterior PVC
monocoil coating 288, PVC shrink tubing 290 over the optical fiber bundle,
and a dual plug body 292. Optical fiber bundle 262 bifurcates into a
reference arm connector 296 and sample arm connector 298, both connected
mechanically by a connector 300. Connector 300 is shown in a side view in
FIG. 9(c). A spacing "d" of about 1.094" (28 mm) is provided between the
center of the reference arm 268 and sample arm 266. The short axis of the
sample fiber bundle is parallel to the long axis of the capillary and
perpendicular to a horizontal line defined between the center points of
the sample and reference arms.
The above described structure is fastened together with 2039 type epoxy. A
360.degree. twist is provided in the fibers in the common sector 288 to
increase flexibility.
In accordance with the invention the cross-section shape of the sample
fiber optic bundle 266 may be varied in accordance with the application.
For instance, in the case where the light beam in the sample arm is to be
transversely focused into a cylinder such as the capillary, it is most
desirable to provide a rectangular cross-sectional shape light beam.
Thereby the fiber optic bundle is provided in a rectangular or
parallelogram shape. In another case when it is desirable to focus the
sample light beam into a cylindrical flow cell as in a liquid
chromatography detector, then it is desirable to have a circular shape of
the cross-section of the light beam and thereby the fiber optics are
bundled into a circle in cross-section.
FIG. 9(d) shows the optical fiber pattern in sample arm 266 in a
rectangular cross sectional arrangement. The overall width w is about 3.05
mm; the height h is about 0.46 mm. Shown are optical fibers a-1, a-2, ...,
a-24.
FIG. 9(e) shows the lower end of the remote optical path, with the beam
splitter common trunk 262 bifurcating into the sample arm 266 and
reference arm 268, both entering beam splitter block 304. Each arm 266,
268 is respectively attached to block 304 by a set screw 306, 308. The
reference photodiode assembly 272 is shown, as is lens shroud 310 to carry
the sample light beam to the sample cell (not shown). Beam splitter block
304 is fastened to platform 314, which is attached to support 316 by a set
screw 320 and a set of dowels 321 (only one shown).
Detail of the sample photodiode assembly is shown in FIG. 9(f). Shown are
the photodiode housing 322, spring assembly outer ring 324, and spring
assembly inner ring 326.
FIG. 10 shows an end-on view of fiber optic bundle 262 showing that the
fiber optic bundle 262 is composed of a number of triads of single optic
fibers. Each triad consists of one reference type fiber 330 (shown by
shading) and two sample type fibers 332, 334 (shown in white). The fibers
themselves are identical between the sample and reference fibers. The
designation of reference or sample merely indicates to which photodetector
the optic fiber directs its light. The fiber triads are arranged in
conjunction with each other so as when one moves from one reference plane
at the entry portion of the beam splitter to another reference plane the
triad is always conserved, so that at any angle the light is introduced to
two sample and one reference optic fibers. Fiber optic bundle 262 in total
includes in one embodiment 37 optic fibers. The diameter of common fiber
optic bundle 262 is preferably about 0.067 inches (1.7 mm). This is a
matter of design choice, and is not limiting in accordance with the
invention. Twice as many sample fibers are provided as reference fibers,
since the sample light beam must pass through the capillary tubing and
other optics and thus there is more loss of throughput in the sample light
beam.
In one embodiment of the invention, the fiber optic bundle is custom made.
The optic fibers are ultraviolet transparent quartz approximately 200
microns in diameter, with a 20 micron thick cladding, and a 12.5 micron
thick polyimid coating. The optical fibers are 200/220/245 superguide G
type. The bundle is supported loosely by a 0.125 inch (3.1 mm) inside
diameter teflon tube in a PVC monocoil outer jacket. The fiber optic
bundle in one embodiment is provided by Highlight Fiber Optics in
Caldwell, Id. The approximate overall length of the beam splitter is 28
inches (70 cm.). The point of bifurcation between the sample arm and the
reference arm is at 26 inches (65 cm.) from the entry portion of the beam
splitter.
Constant Capillary Electrical Resistance Temperature Control
Also in accordance with the invention, constant resistance cooling of the
capillary is provided. As described above, the electrical resistance of
the capillary provides a means of sensing the temperature of the
capillary. Therefore, a method is provided for measuring and controlling
the temperature of the capillary using the apparatus as shown in FIG. 6.
It is well known that the electrical resistance of the capillary is
directly proportional to the capillary length and inversely proportional
to the capillary radius squared. The solution electrical resistance is
inversely proportional to the temperature of the solution and is inversely
related to the specific conductivity of the solution in the capillary.
This means that for a capillary of a given size and a given length
containing a given solution, the electrical resistance is a direct
function of the capillary temperature. In accordance with the invention,
the high voltage power supply's current and voltage conventional sense
lines are used to measure the electrical resistance of the capillary, and
so in effect the capillary is used as a thermometer.
A control procedure is provided to control the temperature of the
capillary. This control procedure is a control program associated with the
above-mentioned microprocessor 142 (see FIG. 7) resident in the
electrophoresis instrument. The procedure for temperature control is shown
in a flow chart in FIG. 11.
In accordance with the invention, the following steps are used in order to
control temperature. First, a voltage start slope is selected at step 340.
(See voltage vs. time plot, FIG. 12.) This is the rate (shown by the
dotted line in FIG. 12) at which the ultimate separation voltage will be
applied. For example, if the electrophoresis separation voltage of 30 KV
is achieved in 10 seconds, then the start slope is 3 KV/second. Second, a
set point ambient temperature is selected at step 342 for the capillary
temperature as desired. This is done by the conventional method of
monitoring the temperature of the air around the capillary tube and
allowing sufficient time at step 344 for the heat transfer process to take
place until the capillary tube approaches the target temperature and
therefore the temperature in the capillary is very close to that of the
surrounding air.
In the next step 346, the electrophoresis separation process in the
capillary begins by performing a sample injection and beginning the run by
increasing at step 348 both the current and the voltage of the electric
power provided to the capillary. During the calibration phase the current
and the voltage are increased at a particular steady rate, equivalent to
two times the start slope at step 350. Capillary resistance is calculated
during the hold time at step 352 (shown as about 0.8 to 2.4 seconds in
FIG. 12) at 5 KV, at which level typically there is no significant joule
heating. In the next step 354 the weighted average resistance or average
resistance for the calibration period hold time is calculated. This
calculated resistance is then attributed to the resistance of the system
at the selected set point temperature. The voltage level is increased to
10 KV at twice the selected start slope in step 356. Then the voltage is
further increased to the set voltage at the selected start slope in step
358.
The next phase in steps 360 to 362 is the temperature control phase. The
resistance is monitored at step 360 at a particular duty cycle, i.e., for
instance 50 times per second, by measuring the capillary current and
voltage, and then in step 362 heat is either pumped into or out of the
chamber in which the air cooled cartridge is housed by use of the
previously described fan and Peltier device. Thus the electrical
resistance of the capillary is maintained at a constant level, providing a
constant temperature.
Buffer Gradient And Temperature Gradient Capillary Electrophoresis
Micellular electrophoretic chromatography is known in the art. (See Terabe,
J. of Microcolumn Techniques, Vol 1, No. 3, 1989, p. 150.) This technique
involves formation of a micell in the sample by providing a buffer
solution containing amphophilic complexes which bind by non-polar or
lipophillic attraction. They remain soluble in aqueous environments due to
their polar moieties. For capillary, micellular electrokinetic
chromatography, typically buffer solutions composed of acid or base salts
(including but not limited to phosphate, tris, hepes, citrate, borate,
amino acids, and other zwitter ionic buffers) in concentrations from 0.01
millimole to 500 millimole are used in conjunction with a detergent or
other lipid-like moiety which forms micells. The micell producing agent
(including but not limited to sodium dodecylsulfate, bile acids, etc.) is
added until reaching minimal micell concentration for the given
temperature.
In accordance with the invention, micellular, open tube separations take
advantage of the differences in the partition coefficients of various
solutes so that the higher the partition coefficient the longer the
solutes stay in contact with the micell under the influence of the
electric field in the electrophoresis instrument. Thus it is possible to
separate neutral compounds on the basis of their partition coefficients.
However, a problem arises in trying to separate solutes of similar
partition coefficients or whose partition coefficients are so large that
they co-migrate on the micells and are never separated. In buffer gradient
electrophoresis, the buffer composition is changed over time and thus
because the basic function of the partition coefficient is dependent on
the two phases, polar and non-polar components (polar component being the
buffer and the non-polar, the micell), the solubility of the solute in the
buffer is changed. Thus as the lipophilicity of the buffer is increased,
those compounds that have slightly lower partition coefficients will come
off the micell. Thus the compounds are selectively removed from the micell
as a function of time and thus contact the detector in the instrument and
are observed.
The gradient micellular chromatography apparatus is depicted in FIG. 13. A
pair of conventional microliter syringe pumps 370, 372 are driven at
different rates to displace different amounts of fluids which when mixed
comprise the buffer. Mixing occurs in a conventional micromixer 376 and
the resultant mixture is transported to separation capillary 378 via
gradient buffer transfer line 380. High voltage electrode 382 creates an
electric field in separation capillary 378.
Fluid from the gradient buffer transfer line 380 enters separation
capillary 378 (see FIG. 14 showing detail of the device of FIG. 13) via
electro-osmotic flow (and not parabolic pressure driven flow) as long as
the pressure head at point P3 is much greater than that at point P2. The
excess buffer exits via waste transfer line 386. Sufficient mixing
response time is achieved using this split- | | |
