Integrated temperature control/alignment system for high performance capillary electrophoretic apparatus

An integrated temperature control/alignment system for use with a high performance capillary electrophoretic apparatus comprises a complementary pair of capillary column mounting plates formed from an electrically insulative, high thermal conductivity material for mounting a capillary column. The first mounting plate includes a well having a predetermined circumference, first and second grooves contiguous with the well and ports formed in the grooves and extending through the plate. The second mounting plate includes a plug configured for insertion in the well and ports aligned with the ports of the first mounting plate. Respective ends of the capillary column are seated and locked in the grooves to preposition the capillary column with respect to the ports. Capillary columns of varying lengths may be mounted within the mounting plates by coiling the intermediate portion thereof within the well to increase or decrease the working length of the column in predetermined increments, depending upon the particular application. Optical coupling elements are disposed in the ports to couple sampling and reference radiation to and from the mounted capillary column. The temperature of substantially the entire capillary column is controlled or regulated by at least one thermopile disposed in thermal contact with one of the mounting plates. A thermistor may be thermally coupled to the capillary column or one of the mounting plates to provide feedback signals to operate the thermopile to maintain the capillary column at a predetermined electrophoretic operating temperature.

FIELD OF THE INVENTION 
This invention relates generally to electrophoretic apparatus, and more 
particularly to a temperature control/alignment system integrated with a 
high performance capillary electrophoretic apparatus. 
BACKGROUND OF THE INVENTION 
Capillary electrophoresis is a technique for analyzing and/or purifying a 
wide variety of biochemical substances or analytes such as proteins, 
nucleic acids, carbohydrates, hormones and vitamins. In particular, 
electrophoresis is an extremely efficacious and powerful means for the 
identification and/or separation of analytes based upon ultra-small volume 
samples. In general, electrophoresis is a phenomenon that involves the 
migration of charged particles or analytes through a conducting liquid 
solution under the influence of an applied voltage. 
The basic capillary electrophoretic apparatus consists of a capillary 
column having the ends thereof positioned in reservoirs containing 
electrodes. A conducting liquid or buffer solution disposed in the 
reservoirs and the capillary column comprises the electrophoretic 
conductive circuit. 
An analyte is injected into the appropriate end of the capillary column and 
a voltage applied across the electrodes. The applied voltage causes the 
analyte to migrate electrophoretically through the capillary column past a 
prepositioned on-column detection device to generate an electropherogram, 
a graphical representation of the analyte. 
Electrophoresis may be conducted in "open" or "gel" capillary columns. Open 
capillary electrophoresis can be conducted either with or without 
electroosmosis which involves bulk solvent migration under the influence 
of the applied voltage as a result of the charged condition of the inner 
wall of the capillary column. Gel capillary electrophoresis, in which the 
interior channel of the capillary column is filled with a suitable gel, 
provides the potential for different modes of separation based upon size 
of the analytes. 
In either open or gel capillary electrophoresis, however, the applied 
voltage is a primary factor affecting the migration of the analyte. 
Therefore, the term electromigration as used herein encompasses either or 
both forms of voltage induced analyte movement. 
An effective high performance capillary electrophoretic system provides 
high resolution, high sensitivity, short run times, on-line monitoring or 
detection of the analyte, and reproducible performance. One practical way 
of enhancing the performance of a capillary electrophoretic apparatus is 
by the application of high applied voltages. Another is to utilize 
shortened capillary columns. Both of these means of enhancing the 
effectiveness of the electrophoretic apparatus, however, have heretofore 
been limited due to the Joule heat generated in the capillary column 
during the electrokinetic separation operation which adversely affects 
electrophoretic separations. 
The applied voltage causes a current flow in the buffer solution of the 
electrophoretic apparatus that is generally defined by Ohm's Law. The 
current flow through the capillary column generates Joule heat or thermal 
energy in the capillary column. Increasing the applied voltage increases 
the current flow and the resulting increased power raises the amount of 
Joule heat generated, which is generally an adverse condition. Similarly, 
shortening the length of the capillary column decreases the capillary 
column resistance, thereby causing an increase in current flow for a given 
applied voltage with the concomitant increase in Joule heating. 
Column temperature influences most of the important physical and chemical 
parameters involved in high performance capillary electrophoresis. In 
particular, column temperature directly affects electrophoretic separation 
since there is a variation in mobility of about 2%/.degree.C. For any 
given electrophoretic separation, there is generally a preferred column 
temperature for optimal separation conditions. A column temperature 
deviation of only 1.degree. C. may affect the migration rate, thereby 
adversely impacting on separation reproducibility. Other adverse effects 
that may result from column temperature deviations include reduced 
separation efficiency, sample decomposition, and the inability to maintain 
the desired chemical equilibria. 
Joule heat generated within the capillary column, if not efficiently 
controlled and/or dissipated to the ambient environment, causes a 
temperature buildup within the column. The temperature buildup 
detrimentally affects the electrophoretic separation by inducing 
variations in column resistance, which affects current flow, and 
concomitantly Joule heating, in the column FIG. 18 illustrates the 
variations in column current with time, with a constant applied voltage, 
for various methods of cooling a capillary electrophoretic column. 
Curves 1 and 2, respectively, illustrate column cooling by natural 
convection and forced air convection with a fan. An examination of these 
curves reveals a noticeable variation in column current with time. Point A 
indicates the effect of the operation of an air conditioner in the 
laboratory, and shows that both natural convection and forced air cooling 
are susceptible to changes in the laboratory environment. Curve 3 
illustrates cooling of the capillary column by means of a solid state 
cooling device according to the present invention. The solid state cooling 
device provides: (1) isolation of capillary column from ambient 
environmental conditions and (2) precise control of the temperature of the 
capillary column. FIG. 18 also graphically illustrates the fact that, 
although each electrophoretic separation was conducted under the same 
operating conditions, there was a significant deviation in column current 
flow, and hence column operating temperature (57.degree. C., 33.degree. 
C., 24.degree. C., respectively, based upon the corresponding column 
current flow and the mobility rate of 2%/.degree.C.), among the 
electrophoretic operations due to the different methods of cooling. 
An optimized high performance capillary electrophoretic apparatus provides 
statistically reproducible results for equivalent analytes, with minimum 
band broadening of the output. Preferably, the apparatus is operated at 
high applied voltages to provide high speed, efficiency and resolution of 
separations. An optimized high performance capillary electrophoretic 
apparatus must include a temperature control system that maximizes 
reproducibility of column resistance and minimizes any detrimental thermal 
effects on separation. The temperature control system, in addition to 
effectively maintaining a constant column temperature, should have the 
capability to vary the column temperature depending upon the particular 
application. 
For example, it may be important to vary column temperature to manipulate 
chemical equilibria such as metal chelation and micelle partitioning. 
Electrophoretic separations below ambient temperature have been shown to 
be useful in minimizing proteolysis or sample decomposition. 
Electrophoretic separation of oligonucleotides, in contrast, have been 
improved by injecting at 60.degree. C. where the species adopts a random 
coil configuration. 
Prior art attempts to cool the capillary electrophoretic apparatus have 
involved natural and forced convection cooling. Due to disadvantages 
associated with many cooling solvents, e.g., low cooling capacity, 
flamability, toxicity, and/or high costs, water has generally been used as 
the cooling element in prior art electrophoretic convection cooling 
systems. In additional to being high capacity devices, requiring two to 
four liters of water, water-cooled devices suffer a marked degradation in 
cooling performance, about 20 to 40 percent, at temperatures approaching 
four degrees centigrade. And while water-cooled devices provide an 
improvement over air-convection devices in controlling column temperature, 
water-cooled devices are severely limited in capability to rapidly vary 
the column temperature for different applications, and a relatively 
expensive. In addition, a water coolant has a sufficient degree of 
electrical conductivity to interfere with the electrophoretic separation 
process. 
Another limiting aspect of prior art capillary electrophoretic apparatus 
was due to the fact that the structural configuration of temperature 
regulating systems severely hindered temperature control in the detection 
zone. A lack of temperature control can lead to non-reproducible results 
in migration rates and separation. Likewise, for accurate collection of a 
given species in micropreparative applications, the column length between 
the detection zone and the collection point should be minimized, and this 
column length should have similar temperature characteristics to the 
separation region prior to the detection zone in order to predict 
accurately when a peak leaves the column. 
Moreover, reproducible results in signal-to-noise ratio were difficult to 
achieve in prior art capillary electrophoretic apparatus due to the 
cumbersome and time consuming effort required to properly align and lock 
the capillary column with respect to the prepositioned detection device. 
Improper alignment of or failure to lock the capillary column in a 
predetermined position leads to the generation of variable noise due to 
vibration effects, which can lead to poor detection limits. 
SUMMARY OF THE INVENTION 
The present invention surmounts the inherent disadvantages of the prior art 
by providing an integrated temperature control/alignment system for a high 
performance capillary electrophoretic apparatus. The integrated 
temperature control/alignment system provides a means for regulating 
and/or varying the temperature of the capillary column, over substantially 
the entire working length thereof, to a predetermined operating 
temperature by dissipating or in some instances augmenting, in order to 
operate at elevated column temperatures, the Joule heat generated by 
current flow through the capillary column. 
Temperature regulation of the capillary column is effected by controlling a 
secondary current flow through a thermoelectric device, thereby regulating 
heat transfer away from or into the capillary column. An effective means 
of temperature regulation permits a higher voltage to be applied across 
the capillary column of given diameter than if the system were not 
present, with the accompanying increase in apparatus performance. The 
structural configuration of the integrated temperature control/alignment 
system also permits thermoelectric temperature regulation over the 
on-column detection zone of the capillary column. 
In addition, the integrated temperature control/alignment system provides a 
means for mounting the capillary column as a constituent of the 
electrophoretic apparatus. The structural configuration of the integrated 
temperature control/alignment system facilitates the precise alignment of 
the detection "windows" of the capillary column with the detection 
openings of the integrated temperature control/alignment system for 
on-column detection. The structural configuration of the integrated 
temperature control/alignment system coacts with the capillary column to 
lock the capillary column therein in a predetermined aligned position with 
respect to the on-column detection device. The locking feature of the 
present invention effectively eliminates adverse vibratory effects which 
would have affected the output while the alignment feature ensures 
reproducible results. 
One exemplary embodiment of the integrated temperature control/alignment 
system according to the present invention comprises a pair of capillary 
column mounting plates for mounting the capillary column as a constituent 
of the electrophoretic apparatus, a pair of secondary support plates, a 
pair of thermoelectric plates and external heat sink plates. 
The capillary column mounting plates are formed from an electrically 
insulating material and each plate has a lengthwise groove sized to snugly 
seat the capillary column. With the capillary column mounting plates 
disposed in contacting relation with the capillary column sandwiched 
therebetween in the grooves, the capillary column is effectively locked 
into position within the integrated temperature control/alignment system. 
A detection slit and a detection hole are formed through the respective 
capillary column mounting plates so as to be substantially centered about 
the lengthwise groove thereof. The capillary column is seated within the 
grooves of the capillary column mounting plates so that the detection 
windows thereof are precisely aligned with the detection slit and hole, 
respectively. This ensures that the capillary column is mounted as a 
constituent of the electrophoretic apparatus in alignment with the 
prepositioned on-column detection device. 
The secondary support plates are fabricated and utilized to facilitate heat 
transfer between the encapsulated capillary column and the thermoelectric 
plates as well as to provide for increased structural strength of the 
integrated temperature control/alignment system. Each secondary support 
plate has a detection slot formed therethrough. The secondary support 
plates are disposed in contact with the exterior facing surfaces of the 
respective capillary column mounting plates in such manner that the 
detection slots are aligned with the detection slit and hole, 
respectively. 
Thermoelectric plates are disposed in contact with the exterior facing 
surfaces of the respective secondary support plates. Each thermoelectric 
plate includes one or more thermoelectric conducting circuits or 
thermopiles functioning to regulate heat transfer with the capillary 
column. During typical electrophoretic operating conditions, the 
thermoelectric plates function to provide thermoelectric cooling for the 
capillary column sandwiched within the integrated temperature 
control/alignment system by transferring Joule heat produced within the 
capillary column through the respective plates of the integrated 
temperature control/alignment system to the ambient environment. 
Controlling the direction and magnitude of the current flow through the 
thermoelectric conducting circuits permits thermoelectric regulation of 
both the direction and rate of heat transfer with the capillary column, 
and the ambient environment as required. The direction of heat transfer 
may be such as to either dissipate or augment the Joule heat generated 
within the capillary column, such that the temperature of the capillary 
column is precisely controlled. Each thermoelectric plate can include a 
detection passageway aligned with the respective detection slot, the 
detection passageway being required when the thermoelectric plate is one 
unitary thermopile, that is a single thermoelectric conducting circuit. 
External heat sink plates are disposed in contact with respective 
thermoelectric plates to transfer Joule heat away from the capillary 
column when the system operates to provide thermoelectric cooling of the 
capillary column. The external heat sink plates act to transfer the 
thermal energy or Joule heat of the higher temperature surface of the 
thermoelectric plates to the external ambient environment. Heat transfer 
to the ambient environment may be effected by radiative, convective, 
and/or conductive heat transfer from the external heat sink plates. 
Detection apertures can be formed through the external heat sink plates so 
as to be aligned with the detection slots or the detection passageways, 
depending upon the particular configuration of the thermoelectric plates. 
Another exemplary embodiment of the integrated temperature 
control/alignment system consists of a module that is configured to 
include means for mounting capillary columns of different length while 
concomitantly providing solid state cooling over substantially the entire 
working length thereof and means for decoupling the detection device from 
the module. The module is configured for slidable insertion and removal 
from an electrophoretic container that provides interfacing with 
associated electrophoretic electronics, detection equipment, power supply, 
and ancillary cooling equipment such as heat sinks and/or forced air 
convection gear. One or more thermopiles are disposed in combination with 
the module to control and/or vary the temperature thereof. 
The module comprises first and second mounting plate members formed from an 
insulative, highly thermal conductive material such as alumina and 
configured to be mated together in combination. The first and second 
mounting plate members, in combination with the associated thermopiles, 
provide solid state temperature control for the capillary column disposed 
therewith. 
The first mounting plate member includes a well and first and second 
grooves extending from diametrically opposed points of the well to an edge 
of the plate. Ports are formed in respective grooves and extending through 
the first plate member for respective elements of the sample and reference 
optical coupling means. The first and second grooves are dimensioned for 
snugly seating the respective ends of a capillary column. The well is 
defined by a circumferential wall having a predetermined circumference. 
The intermediate portion of the mounted capillary column abuts against the 
circumferential wall. 
The well provides the capability to accommodate capillary columns of 
different length such that the module may be utilized to provide varying 
capillary column working lengths. The intermediate portion of polymer 
coated microcapillary columns may be coiled a predetermined number of 
turns within the well to provide a predetermined column working length 
that is variable in increments. The inherent resilience of such columns 
causes the coiled intermediate portion to abut against the circumferential 
wall. 
The second mounting plate member is formed to complement the first mounting 
plate member and includes a plug and ports extending therethrough in 
registration with the ports of the first mounting plate member. The plug 
is configured to be disposed within the well with the second mounting 
block member mated in combination with the first mounting block member. 
Sample and reference optical coupling means such as optical fibers may be 
mounted in corresponding ports of the first and second mounting block 
members. The sample and reference radiation coupling means provide an 
optical interface between the module and the radiation coupling means 
mounted in the container.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Referring now to the drawings wherein like reference numerals designate 
corresponding or similar elements throughout the several views, there is 
shown generally in FIG. 1 a high performance capillary electrophoretic 
(HPCE) apparatus 10 according to the present invention. The basic 
capillary electrophoretic apparatus 10 comprises a capillary tube or 
column 12 having ends 14, 14 disposed in buffer solution reservoirs 16, 
16. 
Electrodes 18, 20 electrically connect the buffer solution reservoirs 16, 
16 to apply a voltage, Vep, across the electrodes 18, 20, respectively, 
thereby providing the motive force for electrophoretic migration of an 
analyte. The capillary column 12 is mounted as a constituent of the HPCE 
apparatus 10 by means of an integrated temperature control/alignment 
system 26, as described hereinbelow in greater detail. 
An on-column detection device 22, 24 is externally prepositioned with 
respect to the HPCE apparatus 10 for on-column detection of the 
electromigrating analyte. For purposes of illustration only, the on-column 
detection device 22, 24 is herein described as a UV radiation source 22 
which focuses UV radiation to pass through the detection openings of the 
integrated temperature control/alignment system 26 and the detection 
windows of the capillary column 12 for ensuing detection by a detection 
means 24. 
The capillary column 12 is a thin-walled, hollow tube, preferably formed of 
a low specific heat, non-conducting material such as fused silica. The 
capillary column 12 may be either "open" or "gel." Typical gels for the 
interior of the capillary column 12 include polyacrylamide or agarose. 
The capillary column 12 typically has a length in the range of 10 cm to 100 
cm, an internal diameter (I.D.) in the range of 25 microns to 200 microns, 
and an outer diameter (O.D.) in the range of 125 microns to 350 microns, 
depending upon the I.D. of the capillary column 12. Capillary columns 
having an I.D. in the range of about 50 to about 100 microns provide 
improved detectability, ease of handling and column loading. 
Since a quartz based fused silica capillary columns 12 having the 
above-disclosed dimensions are relatively fragile, the strength and 
flexibility of the capillary column 12 are generally increased by applying 
an external protective coating of a polymer such as polyimide to the 
capillary column 12. 
The polyimide coating, however, would interfere with the operation of the 
on-column detection device 22, 24. Therefore, the coating of the capillary 
column 12 is typically modified to include detection "windows" which 
permit measurement or sensing of the electromigrating analyte during 
passage through the interior of the capillary column 12. As shown in FIG. 
2, the polyimide coating is selectively removed at opposed locations on 
the capillary column 12 at the predetermined position to create first and 
second detection windows 13, 13, spaced approximately 180 degrees from one 
another. A device for efficiently, selectively and easily removing the 
polyimide coating is described in co-pending application Ser. No. 
07/342,989, filed Apr. 24, 1989, entitled WINDOW BURNER FOR POLYMER COATED 
CAPILLARY COLUMNS. 
Alternatively, a 360 degree peripheral band of the polyimide coating may be 
removed at the predetermined position on the capillary column 12. One of 
ordinary skill in the art will appreciate that "windows" is used herein in 
a generic sense to refer to a modified segment or segments of the 
capillary column 12 at the predetermined position wherein some form of 
measurement/sensing means is provided access into and out of the capillary 
column 12 to detect the electromigrating analyte. To simplify the ensuing 
discussion, the on-column detection device 22, 24 is exemplarily described 
as a UV radiation source and UV detection means. 
The irradiated volume within the capillary column 12 between the detection 
"windows" defines the electrophoretic detection zone. The detection 
radiation generated by the on-column detection device 22, 24 is focused to 
traverse the detection zone of the capillary column 12. 
During the generation of electropherograms the ends 14, 14 of the capillary 
column 12 are disposed in the buffer solution reservoirs 16, 16 in such a 
manner as to be in fluidic contact with the buffer solution. Prior to 
insertion of the ends 14, 14 in the respective buffer solution reservoirs 
16, 16, the interior of the capillary column 12 is filled with the 
gel/buffer solution and the analyte is electrophoretically injected into 
the appropriate end 14 thereof. The buffer solution acts as the 
electrically conductive medium for the electrophoretic circuit. 
The electrophoretic conducting circuit for the HPCE apparatus 10 is 
completed by inserting the first electrode 18 in one buffer solution 
reservoir 16 and the second electrode 20 in the other buffer solution 
reservoir 16. The electrodes 18, 20 are preferably formed of a chemically 
inert material such as platinum to preclude a degrading reaction between 
the electrodes 18, 20 and the buffer solution of the reservoirs 16, 16. 
During electrophoresis, the voltage, Vep, is applied across the HPCE 
apparatus 10 to generate a current in the electrophoretic circuit. The 
applied voltage, V.sub.ep, causes the electromigration of the analyte 
injected into the appropriate end 14 of the capillary column 12 through 
the capillary column 12 to the other end 14. Passage of the analyte 
through the capillary tube 12 past the prepositioned on-column detection 
device 22, 24 causes the generation of an electropherogram which is a 
graphical time display of the quanta of detected radiation. The passage of 
the electromigrating analyte through the detection zone causes the 
monochromatic radiation traversing the capillary column 12 to be scattered 
and/or absorbed. A representative example of an electropherogram derived 
by means of the HPCE apparatus 10 is shown in FIG. 11. 
The exemplary on-column detection device 22, 24 comprises a means for 
generating a monochromatic beam of UV radiation 22 and focusing the beam 
to pass substantially orthogonally through the first detection window 13 
of the capillary column 12. One such monochromatic radiation generating 
means 22 having utility in the HPCE apparatus 10 of the present invention 
is an UV deuterium lamp producing ultraviolet radiation in the 190-380 
nanometer range. By means of a suitable lens/filter arrangement the 
monochromatic beam of ultraviolet radiation, of predetermined wavelength, 
is focused to radiate through the first detection window 13 substantially 
orthogonal thereto. 
The monochromatic radiation traverses the capillary column 12 and the 
medium, e.g., gel, buffer solution and/or analyte solution, disposed 
between the first and second detection windows 13, 13 and exits therefrom 
through the second detection window 13. The exiting radiation is focused 
upon a suitable detection means 24 such as a photomultiplier which is 
responsive to changes in radiation intensity. When the analyte in the 
capillary column 12 electromigrates through the detection zone between the 
first and second detection windows 13, 13 the monochromatic radiation is 
scattered and/or absorbed, thereby generating an electropherogram as shown 
in FIG. 11. 
Other usable monochromatic radiation may be produced by IR and visible 
radiation sources. Fluorescence is another detection technique usable for 
on-column monitoring of the electrophoretically migrating analyte. It will 
be appreciated that the on-column detection technique is not limited to 
the generation and detection of radiation. Other detection techniques 
having utility for on-column detection include conductivity and 
electrochemical measurements/sensing. The common denominator for all 
on-column detection techniques is access to the capillary column in some 
predetermined manner. 
One embodiment of the integrated temperature control/alignment system 26 
for mounting the capillary column 12 as a constituent of the HPCE 
apparatus 10 is shown in greater detail in the end view of FIG. 3. The 
temperature control/alignment system 26 of FIG. 3 is a multi-layered 
structure consisting of a pair of capillary column mounting members or 
plates 28, 28 for mounting the capillary column 12 as part of the HPCE 
apparatus 10, a pair of secondary support plates 30, 30, a pair of 
thermoelectric plates 32, 32, and external heat sink plates 34, 34. 
The capillary column mounting plates 28, 28 illustrated in FIG. 4 are 
preferably fabricated from an electrically insulating material, preferably 
a ceramic-type material having good dielectric characteristics and 
possessing a reasonable coefficient of thermal conductivity. One suitable 
ceramic material is alumina, Al.sub.2 O.sub.3, which has a thermal 
conductivity of approximately 36 W m.sup.-1 .degree.K.sup.-1 at 
293.degree. K. It will be appreciated, however, that the capillary column 
mounting plates 28, 28 may be fabricated metallic conducting materials for 
some applications. Each capillary column mounting plate 28 has a 
lengthwise groove 36 formed in an interior facing surface thereof. 
The groove 36 has a maximum width which is approximately equal to the O.D. 
of the capillary column 12 and a depth which is approximately equal to one 
half of the O.D. of the capillary column 12. The dimensioning of the 
grooves 36 ensures that the capillary column 12 is snugly seated or 
physically engaged therein, and further that when the interior facing 
surfaces of the capillary column mounting plates 28, 28 are disposed in 
contacting relationship, the capillary column 12 is locked or sandwiched 
therebetween in an immotile condition. 
A detection slit 38 is formed through the upper capillary column mounting 
plate 28 in such a manner as to be substantially centered about the groove 
36 thereof and a detection hole 40 is formed through the lower capillary 
column mounting plate 28 in such a manner as to be substantially centered 
about the groove 36 thereof, as shown in FIG. 4. The detection slit 38 has 
a length in the range of about 2 mm or less and a width in the range 
approximately equal to or less than the I.D. of the capillary column 12. 
The detection hole 40 has a diameter in the range of about 2 mm or less. 
When the upper and lower capillary column mounting plates 28, 28 are 
disposed in contacting relationship, as shown in FIG. 3, the detection 
slit 38 is aligned with the detection hole 40. 
The detection slit 38 and the detection hole 40 are formed through the 
upper and lower capillary column mounting plates 28, 28 in such a manner 
that when the capillary column 12 is seated in the opposed grooves 36 of 
the capillary support strips 28, 28, the detection slit 38 can be readily 
aligned with the first detection window 13 and the detection hole 40 can 
be readily aligned with the second detection window 13. The structural 
configuration of the capillary column mounting plates 28, 28 thus 
facilitates the alignment of the capillary column 12 with respect to the 
prepositioned on-column detection device 22, 24, thereby ensuring the 
reproducibility of results as well as consistency therebetween. 
With the grooved faces of the capillary column mounting plates 28, 28 
secured together in a contacting relationship, the capillary column 12 is 
locked in the opposed grooves 36 thereof. A thermally conductive paste may 
be used to ensure a tight, thermally conducting pathway between the 
capillary column 12 and the capillary column mounting plates 28, 28. 
Disposed in contact with the exterior facing surfaces of the capillary 
column mounting plates 28, 28 are the interior facing surfaces of the 
secondary support plates 30, 30, as shown in FIG. 5. The secondary support 
plates 30, 30 are typically fabricated as metal members or plates which 
provide a good thermally conductive medium between the capillary column 
mounting plates 28, 28 and the thermoelectric plates 32, 32 to facilitate 
the transfer of Joule heat between the capillary column 12 and the 
thermoelectric plates 32, 32. 
In addition, the secondary support plates 30, 30 provide increased 
structural strength for the integrated temperature control/alignment 
system 26. Representative metals having utility in forming the secondary 
support plates 30, 30 for use in the present invention include copper, 
which has a thermal conductivity of approximately 398 W m.sup.-1 
.degree.K.sup.-1 at 300.degree. K, and aluminum, which has a thermal 
conductivity of approximately 237 W m.sup.-1 .degree.K.sup.-1 at 
293.degree. K. 
As shown in FIG. 5, a detection slot 42 is formed in each of the secondary 
support plates 30, 30 in such manner that when the integrated temperature 
control/alignment system 26 is assembled in final configuration with the 
capillary column 12 sandwiched therein, the detection slots 42 of the 
secondary support plates 30, 30 are aligned with the detection slit 38 and 
the detection hole 40, respectively, of the capillary column mounting 
plates 28, 28. 
The interior facing surfaces of the thermoelectric plates 32, 32 are 
disposed in contact with the exterior facing surfaces of the secondary 
support plates 30, 30, respectively. Each thermoelectric plate 32 
comprises at least one electrically conducting circuit or thermopile 
utilizing the Peltier effect to thermoelectrically regulate the 
electrophoretic operating temperature, T.sub.cc, of the capillary column 
12 by the controlling the transfer of thermal energy to or from the 
capillary column 12. 
The Peltier effect describes a phenomenon wherein an electric current 
flowing through the junction between two dissimilar metals or conducting 
elements, i.e., a thermocouple, can absorb or generate thermal energy at 
the junction depending upon the direction of the current flow. The rate of 
heat transfer at the junction is directly proportional to the magnitude of 
the current flowing through the junction. The thermal energy transfer rate 
is thus directly controllable by regulating the magnitude of the current 
flowing through the thermoelectric circuit. In addition, by controlling 
the direction of current flow in the thermoelectric circuit, the 
thermoelectric effect exerted on the capillary column 12 is regulated to 
provide either thermoelectric cooling or thermoelectric heating of the 
capillary column 12. 
To simplify the disclosure, the operation of the thermoelectric plates 32, 
32 will be described in terms of providing thermoelectric cooling for the 
capillary column 12, i.e., the thermoelectric plates 32, 32 act to 
transfer the Joule heat generated in the capillary column 12 to the heat 
sink plates 34, 34 via the capillary column mounting plates 28, 28, the 
secondary support plates 30, 30 and the thermoelectric plates 32, 32, 
since electrophoretic operations typically are concerned with the 
dissipation of Joule heat generated within the capillary column 12. The 
thermoelectric cooling provided by the thermoelectric plates 32, 32 thus 
effectively cools the secondary support plates 30, 30, and the capillary 
column mounting plates 28, 28 and concomitantly lowers the electrophoretic 
operating temperature, T.sub.cc, of the capillary column 12 to a 
predetermined operating temperature. 
By connecting a plurality of thermocouples in series to an external current 
source, a thermopile 50 is formed, as schematically illustrated in FIG. 6. 
The thermopile 50 is comprised of a first plurality of conducting elements 
52 and a second plurality of conducting elements 54 joined as shown in 
FIG. 6 to form a first plurality of dissimilar junctions 56 and a second 
plurality of dissimilar junctions 58 electrically connected to a variable 
current source 60. Bismuth telluride is a representative compound used in 
forming the conducting elements of the thermopile 50, with the first and 
second plurality of conducting elements 52, 54 formed by doping the 
bismuth telluride with "p" and "n" type metals, respectively. 
The first plurality of dissimilar junctions 56 are disposed in first planes 
62 to form the interior facing surfaces of the first and second 
thermoelectric plates 32, 32 and the second plurality of dissimilar 
junctions 58 are disposed in second planes 64 to form the exterior facing 
surfaces of the first and second thermoelectric plates 32, 32. The 
junctions may be maintained in such a relationship permanently, as for 
example by fixing the thermocouples in an epoxy infrastructure of suitable 
electrically insulating material. 
Proper selection of the magnitude and direction of the current generated by 
the variable current source 60 flowing in the thermopile circuit 50 
maintains the first plurality of dissimilar junctions 56 at a temperature, 
T.sub.L, while the second plurality of dissimilar junctions 58 are 
maintained at a temperature, T.sub.H, that is, the first plurality of 
dissimilar junctions 56 cool down while the second plurality of dissimilar 
junctions 58 heat up. 
The thermoelectric plates 32, 32 are configured such that during 
electrophoresis the temperature T.sub.L maintained at the first plurality 
of dissimilar junctions 56 is less than the predetermined electrophoretic 
operating temperature, T.sub.cc, at which the capillary column 12 is 
operated during electrophoresis. This ensures that a heat transfer 
potential is maintained between the capillary column 12 and the interior 
facing surfaces of the thermoelectric plates 32,32 such that Joule heat 
generated in the capillary column 12 during electrophoresis is transferred 
away from the capillary column 12. The value of T.sub.L, and hence the 
rate of thermal energy transfer or the thermoelectric cooling effect of 
the thermoelectric plates 32, 32, is regulated by varying the output of 
the current source 60 such that the operating temperature, T.sub.cc, of 
the capillary column 12 is precisely maintained. One or more thermopiles 
50 as described hereinabove are arranged to function singly or in 
combination as the thermoelectric plates 32, 32 utilized in the integrated 
temperature control/alignment system 26 according to the present 
invention. 
The embodiment of the thermoelectric plate 32 shown in FIG. 7 is a unitary 
thermopile 50, that is a single thermoelectric circuit, which has the 
first plurality of dissimilar junctions 56 disposed so as to form the 
interior facing surface 62 in contact with the exterior facing surface of 
the respective secondary support plate 30 while the second plurality of 
dissimilar junctions 58 are disposed to form the exterior facing surface 
64 in contact with the interior facing surface of the respective heat sink 
plate 34. Since the embodiment of the thermoelectric plate 32 of FIG. 7 is 
a unitary thermopile 50, each thermoelectric plate 32 must be modified to 
include a detection passageway 66 aligned with the respective detection 
slot 42 of the corresponding secondary support plate 30 for on-column 
detection of the electrophoretically migrating analyte. 
FIG. 8 illustrates another embodiment of the thermoelectric plate 32 of the 
present invention wherein each thermoelectric plate 32 consists of a 
plurality of individual thermopiles 50, four being shown in FIG. 8, which 
act in combination to transfer thermal energy from the secondary support 
plate 32 to the heat sink plate (not shown). As illustrated in FIG. 8 each 
thermopile 50 has its own current source 60. The individual current 
sources 60 may be gang controlled to regulate the temperature T.sub.L. It 
will be appreciated that the individual thermopiles 50 of each 
thermoelectric plate 32 can be disposed to form, in effect, a detection 
passageway 66. This is accomplished by affixing the individual thermopiles 
50 to the exterior facing surfaces of the corresponding secondary support 
plates 30, 30 so as not to overlap or obstruct the detection slots 42, 42. 
One embodiment of the heat sink plate 34 of the present invention is 
depicted in FIG. 9. Each heat sink plate 34 of the embodiment of FIG. 9 
includes a plurality of radiating fins 70 for externally dissipating the 
thermal energy transferred to the heat sink plate 34 by radiative and/or 
convective heat exchange between the radiating fins 70 and the ambient 
environment where the HPCE apparatus 10 is set up. The convective heat 
exchange of the heat sink plate 34 may be either passive or active. 
Operating the heat sink plate 34 for active convective heat exchange 
requires the utilization of any conventionally known means for forcing a 
greater fluid volume over the surface area of the heat sink plate 34 to 
increase the rate of convective heat exchange between the heat sink plate 
34 and the fluid. Each heat sink plate 34 also includes a detection 
aperture 72 aligned with the respective detection passageway 66 of the 
thermoelectric plate 32 (for the embodiment disclosed by FIG. 7) or the 
detection slot 42 of the respective secondary support plate 30 (for the 
embodiment disclosed by FIG. 8). 
Alternatively, as shown in FIG. 10 the heat sink plate 34 can be primarily 
cooled by conduction by means of a cooling system 74 which circulates a 
cooling fluid for heat exchange through the heat sink plate 34. Such an 
embodiment is effective where the capillary column 12 of the HPCE 
apparatus 10 is to be operated at predetermined operating temperatures 
T.sub.cc which require large quanta of Joule heat to be transferred to the 
ambient environment. The circulating cooling fluid ensures that the 
necessary heat transfer potential is maintained for effective Joule heat 
transfer between the thermoelectric plates 32, 32 and the heat sink plates 
34, 34. This embodiment likewise includes the detection aperture 72 as 
described hereinabove. 
The integrated temperature control/alignment system 26 described 
hereinabove provides an improved means for regulating heat transfer 
between the capillary column 12 and the ambient environment. The direction 
and rate of heat transfer with respect to the capillary column 12 is 
regulated by the thermoelectric effect provided by means of the magnitude 
and direction of the electric current flowing in the thermoelectric plates 
32, 32. Regulation of the direction and rate of heat transfer regulates or 
controls the electrophoretic operating temperature, T.sub.cc, of the 
capillary column 12. By utilizing the integrated temperature 
control/alignment system 26 to thermoelectrically cool the capillary 
column 12 as described in the preceding paragraphs, the HPCE apparatus 10 
according to the present invention is operable at higher V.sub.ep s, which 
results in the generation of higher resolution electropherograms and/or 
analyte separation in shorter run times. By utilizing the integrated 
temperature control/alignment system 26 according to the present 
invention, reproducible high quality results are obtainable down to 
electrophoretic operating temperature, T.sub.cc, of -20.degree. C. 
It is to be understood that the integrated temperature control/alignment 
system 26 of the present invention can also be utilized to 
thermoelectrically heat the capillary column 12, that is, to augment the 
Joule heat generated within the capillary column 12 to raise the 
electrophoretic operating temperature, T.sub.cc,. When the integrated 
temperature control/alignment system 26 is operated to thermoelectrically 
heat the capillary column 12, the heat sink plates 34, 34 need not be 
included as elements of the integrated temperature control/alignment 
system 26. 
It will further be appreciated that the structural configuration of the 
integrated temperature control/alignment system 26 as described 
hereinabove provides thermoelectric regulation of the temperature proximal 
the detection zone of the capillary column 12. This likewise increases the 
accuracy and reproducibility of the electrophoretic separation. Moreover, 
the disclosed structural configuration of the integrated temperature 
control/alignment 26 system facilitates the proper alignment of the 
windows of the capillary column 12 with respect to the prepositioned 
on-column detection device 22, 24, i.e., in the detection zone. 
Other embodiments based upon the integrated temperature/control alignment 
system are possible in light of the above teachings. For example, another 
embodiment of the integrated temperature control/alignment system 
comprises only the capillary column mounting plates and the thermoelectric 
plates as described hereinabove. Another embodiment of the integrated 
temperature control/alignment system comprises only the thermoelectric 
plates, the interior facing surfaces of each thermoelectric plate having 
formed therein grooves and a detection slit and a detection hole, 
respectively, as described hereinabove for seating and locking the 
capillary column in alignment with the prepositioned on-column detection 
device. 
Although the embodiments of the integrated temperature control/alignment 
system have been described hereinabove in terms of a plate configuration, 
it is to be understood that the present invention is not to be limited by 
the terminology "plate". The integrated temperature control/alignment 
system can also be formed as series of individual concentric tubes, as 
functionally described hereinabove, coaxially disposed about the capillary 
column. It will be appreciated that for this embodiment no groove is 
necessary in the capillary column support tube in that the configuration 
of the tube itself performs the seating and locking function. 
Another embodiment of an integrated temperature control/alignment module 
26' for a HPCE apparatus 10 is illustrated in FIGS. 12-16 and includes 
features that permit the module 26' to be utilized under a variety of 
separation conditions. The integrated temperature control/alignment module 
26' includes means for mounting capillary columns of different length 
without changing the basic structural configuration of the module 26' 
while concomitantly providing solid state cooling over substantially the 
entire working length thereof, and means for decoupling the on-column 
detection device 22, 24 from the module 26', as described hereinbelow in 
greater detail. 
A schematic diagram of the HPCE apparatus 10 is illustrated in FIG. 12. The 
apparatus 10 includes a light-tight insulative container 11', a capillary 
column 12' having ends 14', 14' disposed in buffer solution reservoirs 
16', 16'. Electrodes 18', 20' are disposed in the reservoirs 16', 16' and 
electrically connected to a power supply PS to apply a voltage V.sub.ep 
across the electrodes 18', 20'. The capillary column 12' is mounted within 
the container 11' of the HPCE apparatus 10 by means of the integrated 
temperature control/alignment module 26', described hereinbelow in greater 
detail, which is disposed within an insulated holder 100. 
The exemplary on-column detection device consists of a UV radiation source 
22', a detection means 24' and radiation coupling means 80 for coupling 
radiation from the source 22' to the capillary column 12' and from the 
capillary column 12 to the detection means 24'. The capillary column 12' 
is mounted within the integrated temperature control/alignment module 26'. 
The module 26' includes complementary first and second mounting plate 
members 28', 29' and one or more thermopiles 50' mounted in combination 
therewith for controlling and/or varying the temperature of the module 
26'. A thermally conductive substance, for example a high temperature 
thermally conductive paste (Omega Engineering, Inc., Stamford, Conn.) or 
ethylene glycol, may be utilized to enhance the thermal contact between 
the module 26' and the thermopiles 50'. The thermopiles 50 are 
electrically connected to a thermoelectric temperature controller 60'. 
External heat sink plates 34' (FIG. 13) and a cooling system 74', such as 
a fan, may be utilized to effect heat removal from the thermopiles 50'. 
The HPCE apparatus 10 includes a light-tight container 11' that may be 
fabricated from any suitable material such as plexiglass. The container 
11' is configured to receive therein the insulated holder 100. The 
container 11' insulates the operator and module 26' from high voltages. 
The container 11' also minimizes stray light that may affect detection 
results. The container 11' also serves as a support for the insulated 
holder 100, the buffer reservoirs 16', 16', the electrical connections to 
the power supply PS, the external heat sink plates 34' if utilized, and 
the radiation coupling means 80. 
The holder 100, see FIG. 13, is formed from an electrically insulative 
material and is configured so that the integrated temperature 
control/alignment module 26' may be slidably inserted therein or withdrawn 
therefrom. Other features of the holder 100 are described in conjunction 
with other elements in further detail hereinbelow. 
The radiation coupling means 80 includes source fiber optics 82 mounted in 
the holder 100 as shown in FIG. 13 and operative to transmit UV radiation 
from the radiation source to the integrated temperature control/alignment 
module 26'. The radiation coupling means 80 further includes sample cell 
fiber optics 84 mounted in the holder 100 in registration with the source 
fiber optics 82 and operative to transmit radiation from the module 26' to 
the detection means 24'. 
Sample optical coupling means 86 as exemplarily illustrated in FIG. 14 may 
be mounted in combination with the integrated temperature 
control/alignment module 26' to couple radiation from the source fiber 
optics 82 onto the capillary column 12 and to couple radiation exiting the 
capillary column 12' to the sample cell fiber optics 84. In one preferred 
embodiment illustrated in FIG. 14, the sample optical coupling means 86 
comprises two 100 micron optical fibers 86a, a fused silica lens 86b, and 
one 600 micron optical fiber 86c. In another preferred embodiment, the 
sample optical coupling means 86 comprises a first 600 micron optical 
fiber 86a, a fused silica lens 86b, and a second 600 micron optical fiber 
86c. 
The two optical fibers 86a, having lengths of about 30, are secured 
together with cyanoacrylate glue, polished and press-fitted into a teflon 
tube 110 mounted in the first mounting block member 28'. First ends of the 
fibers 86a optically interface with the fused silica lens 86b that focuses 
radiation from the source fiber optics 82 into the fibers 86a. Signal 
losses for the fiber optics 86a above 220 nm are about 1.0 db/m, i.e., 
greater than 80% transmittance. The use of two optical fibers improves the 
signal to noise ratio. For this preferred embodiment, a capillary window 
13' having an axial length of about 240 micrometers is formed in the 
capillary column 12'. 
The 600 micrometer optical fiber 86c is press-fitted into the second 
mounting block member 29'. The 600 micrometer optical fiber 86c optically 
couples radiation exiting the capillary column 12' into the sample cell 
fiber optics 84. 
The HPCE apparatus 10 may also include reference radiation coupling means 
90 comprising first and second reference cell fiber optics 92, 94 and 
reference optical coupling means 96. The reference cell fiber optics 92, 
94 are mounted in registration in the holder 100 and operative to couple 
radiation between the on-column detection device 22', 24' and the 
integrated temperature control/alignment module 26'. The reference optical 
coupling means 96, which may be similar to that described in the preceding 
paragraphs, is disposed in combination with the integrated temperature 
control/alignment module 26' to couple radiation from the first reference 
cell fiber optics 92 onto the capillary column 12' and to couple radiation 
exiting the capillary column 12' to the second reference cell fiber optics 
94. 
The reference radiation coupling means 90 may be utilized to generate an 
electropherogram of the injected analyte. This permits a quantitative 
determination of how much analyte has been injected into the capillary 
column 12'. The above-described configuration provides optical decoupling 
between the fiber optics mounted in the holder 100 and the optical 
coupling means disposed in combination with the integrated temperature 
control/alignment module 26'. 
The complementary mounting plate members 28', 29' are fabricated from a 
material that has a high thermal conductivity and good electrical 
insulating characteristics. Alumina, Al.sub.2 O.sub.3, which has a thermal 
conductivity of about 36 W m.sup.-1 .degree.K.sup.-1, is one material 
having utility for fabricating the mounting plates 28', 29'. Boron 
nitride, Bn, which has a thermal conductivity of about 56 W m.sup.-1 
.degree.K.sup.-1, is another material having utility for fabricating the 
mounting plates 28', 29'. Bn, in addition to having a higher thermal 
conductivity, also is easier to machine than Al.sub.2 O.sub.3, thereby 
reducing fabrication costs. The high thermal conductivity of the plates 
28', 29' provides the solid state cooling capacity that maintains the 
outer surface of substantially the entire length of the capillary column 
12' at the predetermined electrophoretic operating temperature T.sub.cc. 
The plates 28', 29' are configured to be mated in combination and the 
combination is dimensioned to be slidably received in the holder 100. The 
thermal conductivity between the mated plates 28', 29' may be enhanced by 
spreading ethylene glycol on the abutting surfaces of the plates 28', 29' 
prior to mating. The plates 28', 29' are machined to provide good thermal 
contact with and to seat and lock the capillary column 12' within the 
module 26'. With the plates 28', 29' mated in combination with the 
capillary column 12 mounted therein and the ends 14', 14' thereof disposed 
in reservoirs 16', 16' such as 0.5 mL micro test tubes, only about 0.5 cm 
of each end 14' of the capillary column 12' is exposed between the 
reservoir 16' and the integrated temperature control/alignment module 26' 
when mounted in the holder 100. The minimal length of exposed capillary 
column minimizes temperature deviations within the capillary column due to 
Joule heating. 
The first mounting plate 28' includes first and second capillary support 
grooves 120, 122 and a well 124 to support capillary columns of different 
lengths therein. The capillary support grooves 120, 122 are dimensioned 
for snugly mounting the respective ends of a given capillary column. Ports 
126, as required, are formed in the respective support grooves 120, 122 
and extending through the plate 28' for receiving corresponding elements 
of the sample and reference optical coupling means as described 
hereinabove. In one preferred embodiment, exemplarily illustrated in FIG. 
15., a thin (about one ten-thousandth of an inch thick) stainless steel 
mounting member 125 is disposed in the plate 28' about the port 126 
defining the detection zone. The mounting member 125 includes a capillary 
mounting groove 125aand a slit 125b. 
In one preferred embodiment, the capillary support grooves 120, 122 are 
dimensioned for mounting a capillary column 12' having an O.D. of about 
357 micrometers. The I.D. of the capillary column 12' is selectively 
variable, e.g., 50, 100 or 200 micrometers, depending upon the particular 
application. 
The well 124 of the first mounting plate 28' is defined by a 
circumferential wall 124a having a predetermined circumference. Polyimide 
coated capillary columns have sufficient resiliency such that the 
intermediate portion of the mounted column 12' abuts against the 
circumferential wall 124a. The intermediate portion of any particular 
capillary column may be coiled a predetermined number of turns within the 
well 124 so that the capillary column 12' mounted within the integrated 
temperature control/alignment module 26' has a predetermined working 
length. The predetermined working length is determined by the number of 
turns of the intermediate portion of the capillary column and the 
predetermined circumference of the well 124. 
The basic configuration of the integrated temperature control/alignment 
module 26' embodiment, i.e., the well 124, permits capillary columns of 
different lengths to be mounted therein while concomitantly providing the 
same degree of solid state cooling, temperature control, therefor. Thus, 
the integrated temperature control/alignment module 26' may be utilized 
for the electrophoretic separation of a variety of analytes and with gel 
or open columns. 
In one preferred embodiment, the well 124 has a predetermined circumference 
such that capillary columns of different lengths may be coiled therein to 
provide column working lengths of 15, 30, 45 or 60 centimeters, 
respectively. Column working lengths of 45 and 60 centimeters are 
typically used in electrophoretic separations. 
The second mounting plate member 29' is formed to complement the first 
mounting plate 28' and includes a plug 130 and ports 132. The plug 130 is 
configured to be disposed within the well 124 to retain the intermediate 
portion of the capillary column 12' in abutting relation against the 
circumferential wall 124a. The plug 130 may be configured so that a 
predetermined gap, e.g., about 400 microns, exists between the periphery 
of the plug 130 and the circumferential wall 124a so that the capillary 
column 12' may be coated with a high temperature thermally conductive 
paste or ethylene glycol to enhance thermal contact between the column 12' 
and the integrated temperature control/alignment module 26'. 
The ports 132 are formed to extend through the second mounting plate 29' in 
registration with the ports 126 of the first mounting plate 28'. The ports 
132 are configured to receive corresponding elements of the sample and 
reference optical coupling means as described hereinabove. 
Alternative embodiments for the first and second mounting block members 
28', 29' are illustrated in FIGS. 16 and 17. Grooves 127 may be formed in 
the circumferential wall 124a to receive the intermediate portion of the 
capillary column. Grooves 133 may be formed in the sidewall of the plug 
130 to receive the intermediate portion of the capillary column. 
The holder 100 is configured so that a temperature measuring means 140 such 
as a thermistor, thermocouple or RTD may be thermally coupled to the 
integrated temperature control/alignment module 26' to monitor the 
predetermined electrophoretic operating temperature T.sub.cc of the 
capillary column. The first and second mounting plates 28', 29' may be 
further configured so that the temperature measuring means 140 may be 
disposed in direct thermal contact with the capillary column 12'. The 
temperature measuring means 140 is electrically coupled to the 
thermoelectric temperature controller 60' to provide temperature feedback 
signals thereto to facilitate temperature control of the capillary column. 
The use of the temperature measuring means 140 enhances the stability of 
the solid state temperature control system by providing proportional 
current control. Feedback signals from the temperature measuring means 140 
is fed to the controller 60' to regulate the current flow to the 
thermopiles 50'. This, in turn, regulates the heat transfer rate between 
the module 26' and the mounted capillary column 12'. 
In another embodiment of the present invention, the hereinabove-described 
device may be utilized with an off-line detector such as a mass 
spectrometer or an electrochemical detector In this embodiment, the 
primary function of the integrated temperature control/alignment module 
26' is to provide thermoelectric temperature control for the capillary 
column to maintain same at the predetermined electrophoretic operating 
temperature T.sub.cc. The integrated temperature control/alignment module 
26' is operative for mounting the capillary column 12' and, in addition, 
serves to align the capillary column 12' in the sense that capillary 
column 12' is centered within the module 26', i.e., that is, to ensure 
that the ends 14', 14' of the capillary column evenly extend outwardly 
from the module 26'. 
Other embodiments and variations of the present invention are possible in 
light of the above teachings. It is therefore to be understood that within 
the scope of the appended claims, the present invention may be practiced 
otherwise than as specifically described hereinabove.