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Claims  |
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What is claimed is:
1. A means for capillary zone electrophoresis with laser-induced indirect
fluorescence detection comprising:
a capillary zone electrophoresis system including a capillary tube means
having an anode and a cathode end, power means for imposing an electrical
field upon the capillary tube means, specimen means for supplying specimen
to the capillary tube means, buffer means for supplying buffer to the
capillary tube means;
an indirect fluorescence detection system including a laser means for
directing a laser beam upon a selected portion of the capillary tube
means, a detector means for detecting fluorescence indirectly from the
selected portion of the capillary tube means, the buffer containing as a
principal component a fluorescing portion which fluoresces upon imposition
of the laser beam and which allows indirect fluorescence detection by
causing one of the set of displacement and ion-pairing of the sample and
the electrophoresis portion of the buffer without labeling the sample with
an electrophoretic portion, the detector measuring a signal independent of
the spectral properties of the sample but related to fluorescing changes
caused by the displacement or ion-pairing which indirectly allows
sensitive detection of constituent components of the specimen during the
electrophoresis process; and
an interlock and timing means associated with the power means for
controlling the periods of time the electrical field is created.
2. The means of claim 22 wherein the interlock and timing means includes
safety means for disconnecting electrical power from the system.
3. A means for capillary zone electrophoresis with laser-induced indirect
fluorescence detection comprising:
a capillary zone electrophoresis system including a capillary tube means
having an anode and a cathode end, power means for imposing an electrical
field upon the capillary tube means, specimen means for supplying specimen
to the capillary tube means, buffer means for supplying buffer to the
capillary tube means;
an indirect fluorescence detection system including a laser means for
directing a laser beam upon a selected portion of the capillary tube
means, a detector means for detecting fluorescence indirectly from the
selected portion of the capillary tube means, the buffer containing as a
principal component a fluorescing portion which fluoresces upon imposition
of the laser beam and which allows indirect fluorescence detection by
causing one of the set of displacement and ion-pairing of the sample and
the electrophoresis portion of the buffer without labeling the sample with
an electrophoretic portion, the detector measuring a signal independent of
the spectral properties of the sample but related to fluorescing changes
caused by the displacement or ion-pairing which indirectly allows
sensitive detection of constituent components of the specimen during the
electrophoresis process; and a shunting resistor means electrically
connected between the power means and the anode end of a capillary tube
means.
4. A means for capillary zone electrophoresis with laser induced indirect
fluorescence detection comprising:
a capillary zone electrophoresis system including a capillary tube means
having an anode and a cathode end, power means for imposing an electrical
field upon the capillary tube means, specimen means for supplying specimen
to the capillary tube means, buffer means for supplying buffer to the
capillary tube means;
an indirect fluorescence detection system including a laser means for
directing a laser beam upon a selected portion of the capillary tube
means, a detector means for detecting fluorescence indirectly from the
selected portion of the capillary tube means, the buffer containing as a
principal component a fluorescing portion which fluoresces upon imposition
of the laser beam and which allows indirect fluorescence detection by
causing one of the set of displacement and ion-pairing of the sample and
the electrophoresis portion of the buffer without labeling the sample with
an electrophoretic portion, the detector measuring a signal independent of
the spectral properties of the sample but related to fluorescing changes
caused by the displacement or ion-pairing which indirectly allows
sensitive detection of constituent components of the specimen during the
electrophoresis process; and a power stabilizing means positioned between
the laser means and the capillary tube means for stabilizing the laser
beam.
5. A means for capillary zone electrophoresis with laser induced indirect
fluorescence detection comprising:
a capillary zone electrophoresis system including a capillary tube means
having an anode and a cathode end, power means for imposing an electrical
field upon the capillary tube means, specimen means for supplying specimen
to the capillary tube means, buffer means for supplying buffer to the
capillary tube means;
an indirect fluorescence detection system including a laser means for
directing a laser beam upon a selected portion of the capillary tube
means, a detector means for detecting fluorescence indirectly from the
selected portion of the capillary tube means, the buffer containing as a
principal component a fluorescing portion which fluoresces upon imposition
of the laser beam and which allows indirect fluorescence detection by
causing one of the set of displacement and ion-pairing of the sample and
the electrophoresis portion of the buffer without labeling the sample with
an electrophoretic portion, the detector measuring a signal independent of
the spectral properties of the sample but related to fluorescing changes
caused by the displacement or ion-pairing which indirectly allows
sensitive detection of constituent components of the specimen during the
electrophoresis process; and an imaging means between the capillary tube
means and the detector means for collecting an imaging fluorescence upon
the detector means emanating from capillary tube means where the laser
means has been directed.
6. A means for capillary zone electrophoresis with laser induced indirect
fluorescence detection comprising:
a capillary zone electrophoresis system including a capillary tube means
having an anode and a cathode end, power means for imposing an electrical
field upon the capillary tube means, specimen means for supplying specimen
to the capillary tube means, buffer means for supplying buffer to the
capillary tube means;
an indirect fluorescence detection system including a laser means for
directing a laser beam upon a selected portion of the capillary tube
means, a detector means for detecting fluorescence indirectly from the
selected portion of the capillary tube means, the buffer containing as a
principal component a fluorescing portion which fluoresces upon imposition
of the laser beam and which allows indirect fluorescence detection by
causing one of the set of displacement and ion-pairing of the sample and
the electrophoresis portion of the buffer without labeling the sample with
an electrophoretic portion, the detector measuring a signal independent of
the spectral properties of the sample but related to fluorescing changes
caused by the displacement or ion-pairing which directly allows sensitive
detection of constituent components of the specimen during the
electrophoresis process; and a filtering means in position between the
capillary tube means and the detector means, the filtering means including
a spatial filter.
7. A means for capillary zone electrophoresis with laser induced indirect
fluorescence detection comprising:
a capillary zone electrophoresis system including a capillary tube means
having an anode and a cathode end, power means for imposing an electrical
field upon the capillary tube means, specimen means for supplying specimen
to the capillary tube means, buffer means for supplying buffer to the
capillary tube means;
an indirect fluorescence detection system including a laser means for
directing a laser beam upon a selected portion of the capillary tube
means, a detector means for detecting fluorescence indirectly from the
selected portion of the capillary tube means, the buffer containing as a
principal component a fluorescing portion which fluoresces upon imposition
of the laser beam and which allows indirect fluorescence detection by
causing one of the set of displacement and ion-pairing of the sample and
the electrophoresis portion of the buffer without labeling the sample with
an electrophoretic portion, the detector measuring a signal independent of
the spectral properties of the sample but related to fluorescing changes
caused by the displacement or ion-pairing which indirectly allows
sensitive detection of constituent components of the specimen during the
electrophoresis process; and
a filtering means in position between the capillary tube means and the
detector means, the filtering means including an interference filter for
selecting desired fluorescence wavelengths to be passed to the detector
means.
8. A method of improving the sensitivity of separation and detection in
capillary zone electrophoresis with laser-induced indirect fluorescence
detection comprising the steps of:
mixing an electrophoretic solution with a fluorescent buffer solution to
present a mixture;
presenting a mixture to a capillary tube of a capillary zone
electrophoresis system;
directing a laser beam to a portion of the capillary tube, the laser beam
being stabilized before being directed to a portion of the capillary tube;
indirectly detecting the fluorescence from the mixture passing through
portions of the capillary tube upon which the laser beam is imposed by
measuring a signal independent of the spectral properties of the mixture
but related to the change in fluorescence of the mixture relating to
displacement or ion pairing of the electrophoretic solution and the
fluorescent buffer solution; and
detecting with high sensitivity, the constituent components of the
electrophoretic solution by utilizing the indirect fluorescing
measurements and without labeling the electrophoretic solution with a
fluorescing portion.
9. A method of improving the sensitivity of separation and detection in
capillary zone electrophoresis with laser-induced indirect fluorescence
detection comprising the steps of:
mixing an electrophoretic solution with a fluorescent buffer solution to
present a mixture;
presenting a mixture to a capillary tube of a capillary zone
electrophoresis system;
directing a laser beam to a portion of the capillary tube, the laser beam
being stabilized before being directed to a portion of the capillary tube;
indirectly detecting the fluorescence from the mixture passing through
portions of the capillary tube upon which the laser beam is imposed by
measuring a signal independent of the spectral properties of the mixture
but related to the change in fluorescence of the mixture relating to
displacement or ion pairing of the electrophoretic solution and the
fluorescent buffer solution;
detecting with high sensitivity, the constituent components of the
electrophoretic solution by utilizing the indirect fluorescing
measurements and without labeling the electrophoretic solution with a
fluorescing portion; and
the laser-induced fluorescence from the mixture being filtered spatially
before detection.
10. A method of improving the sensitivity of separation and detection in
capillary zone electrophoresis with laser-induced indirect fluorescence
detection comprising the steps of:
mixing an electrophoretic solution with a fluorescent buffer solution to
present a mixture;
presenting a mixture to a capillary tube of a capillary zone
electrophoresis system;
directing a laser beam to a portion of the capillary tube, the laser beam
being stabilized before being directed to a portion of the capillary tube;
indirectly detecting the fluorescence from the mixture passing through
portions of the capillary tube upon which the laser beam is imposed by
measuring a signal independent of the spectral properties of the mixture
but related to the change in fluorescence of the mixture relating to
displacement or ion pairing of the electrophoretic solution and the
fluorescent buffer solution;
detecting with high sensitivity, the constituent components of the
electrophoretic solution by utilizing the indirect fluorescing
measurements and without labeling the electrophoretic solution with a
fluorescing portion; and
the laser-induced fluorescence of the mixture being interface filtered
before detection. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
a. Field of the Invention
The present invention relates to analytical procedures for detection and
determination of chemical constituents of substances, and particularly to
separation and detection of very low levels of chemical compounds.
b. Problems in the Art
Many analytical procedures exist with respect to attempting to determine
the chemical makeup of substances. For example, procedures such as
chromotography, electrophoresis, fluorescence detection, and others have
been developed for this purpose. Each of those broad categories, in turn,
have many different variations.
Each of these methods has its strengths and weaknesses. For example, one
type of electrophoresis, namely capillary zone electrophoresis, has very
good separation efficiency, for separating out different chemical
components, but is limited in its ability to detect what those components
are. In many cases, to accurately detect the constituent chemical
compounds, additional procedures are required which are very time
consuming, and some of which require and result in destruction or
alteration of the substance being analyzed.
While many of these analytical methods give what are many times considered
acceptable results, these results are many times limited to a selected or
narrow group or type of substances. Therefore, problems exist in that
there is no adequate universal-type procedure which can separate and
detect a wide variety of substances.
Further problems exist with conventional analytical methods with regard to
the amount of time required for resolution and derivation of meaningful
information, and also with respect to the reliability of the information.
Some detection procedures affect the separation process, dilute the
sample, or otherwise bring into doubt the reliability of the entire
separation and detection process.
Furthermore, with conventional procedures, there are significant problems
with respect to getting efficient and accurate information regarding small
amounts of materials to be analyzed, or in analyzing materials having
small fractional amounts of chemical compounds. This is especially true
for materials which have constituent chemical compounds which do not have
inherent physical properties such as UV (ultraviolet) or visible
absorption, fluorescence, or electrochemical characteristics.
Thus, a primary problem exists for analyzing substances having constituent
chemical compounds which are not significantly fluorescing. Such
substances are quite abundant in biotechnological and biochemical areas,
which are of particular interest.
There is therefore a real need for an improvement in the art with respect
to the problems discussed above. There is a need for an analytical
procedure which is more efficient than conventional procedures with regard
to time and derivation of results, and which improves the efficiency of
separation and detection of chemical constituent compounds. Additionally,
there is a need for a procedure that is flexible and applicable to many
different types of substances and situations so that it can be used
somewhat universally. There is also the need for a procedure which can
function efficiently and reliably with regard to small sample amounts or
with regard to samples having minute fractional amounts of compounds which
are either difficult or impossible to detect by conventional methods.
It is therefore a principal object of the present invention to provide a
means and method of capillary zone electrophoresis with laser-induced
indirect fluorescence detection which improves over or solves the
deficiencies and problems in the art.
A further object of the present invention is to provide a means and method
as above described which is reliable and efficient.
A further object of the present invention is to provide a means and method
as above described which is fairly universal in its application, yet
simple in procedure and in apparatus to accomplish the procedure.
Another object of the present invention is to provide a means and method as
above described which is non-destructive, and does not alter the
characteristics of the substances being analyzed.
A further object of the present invention is to provide a means and method
as above described which saves significant time in deriving results.
Another object of the present invention is to provide a means and method as
above described which is operable with respect to very small quantities of
sample materials to be analyzed.
Another object of the present invention is to provide a means and method as
above described which is reliable with respect to detectibility of minute
fractional amounts of chemical compounds.
A further object of the present invention is to provide a means and method
as above described which improves and makes more efficient the separation
of chemical compounds, the detection of chemical compounds, and the
sensitivity to detection of chemical compounds.
A still further object of the present invention is to provide a means and
method which is useful for a variety of analytical situations and
substances.
Another object of the present invention is to provide a means and method as
above described which is useful for non-fluorescing substances.
Another object of the present invention is to provide a means and method as
above described which is safe and economical.
These and other objects, features, and advantages of the present invention
will become more apparent with reference to the accompanying specification
and claims.
SUMMARY OF THE INVENTION
The present invention presents a combination of capillary zone
electrophoresis with laser induced indirect fluorescence detection to
provide an improvement in the art with regard to separation and detection
of chemical components of analyzed substances. An open capillary zone
electrophoresis system includes a detector which operates to indirectly
measure changes in fluorescence induced by the laser. An output from the
detector means can be communicated to a data processing device or a
recording means to make a record of such detection correlated to time, and
to derive meaningful results.
The invention represents a universal, simple, and efficient analytical
procedure, which improves upon the amount of time needed, the separation
efficiency, and the detection sensitivity, presenting a reliable and
economical analytical tool.
The invention includes various features and options which enhance its
results and make it particularly advantageous for reliably deriving
information from very small amounts of sample material, and for detecting
very small fractional amounts of chemical compounds. Its high sensitivity
and universality make it also particularly useful with regard to
biochemically important molecules.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagrammatic view of an analytical system according to the
system.
FIG. 2 is a diagrammatic view of the detector of FIG. 1.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention has been described generally above. To assist in an
understanding of the invention, a preferred embodiment of the invention
will now be described in detail. Reference should be taken to the
drawings, namely FIGS. 1 and 2. Reference numerals will be used to
designate various components and features in the drawings. Like reference
numbers will be used for like parts in each of the Figures.
With particular reference to FIG. 1, a diagrammatic view of preferred
embodiment 10 of the invention is shown. A capillary zone electrophoresis
system consisting of a power section 12, and a capillary section 14, such
as is well known in the art, is utilized. For specifics regarding the
components and operation of capillary zone electrophoresis systems,
reference is given to Jorgenson and Lukacs, "Capillary Zone
Electrophoresis" Science Vol. 222, pgs. 266-272, which is hereby
incorporated by reference.
Power section 12 selectively introduces high voltage electricity through
capillary section 14. A buffer solution 16 is put in fluid communication
with capillary tube 18. A sample or specimen material to be analyzed is
injected into one of the open ends of capillary tube 18. Upon application
of the high voltage, the sample separates by migrating through the
capillary tube 18 according to the reaction of different constituent
compounds to the electric field. In other words, ionic components of the
specimen or sample migrate through the capillary tube 18 at different
speeds based upon their particular electrical characteristics.
Thus, the capillary zone electrophoresis system presents an efficient way
of separating constituent chemical compounds of the sample.
A detector 20 is added to the capillary zone electrophoresis system
according to the invention. Detector 20 utilizes indirect fluorescence
detection principles to derive information and detect the constituent
chemical compounds separated by the electrophoresis system as they pass by
detector 20. Indirect fluorescent detection is known within the art and
one type of this sort of analytical procedure is explained in Mho and
Yeung, "Detection Method for Ion Chromotography Based on Double-Beam
Laser-Excited Indirect Fluorometry", Analytical Chemistry, 1985, 57,
2253-2256, which is incorporated by reference herein.
It is to be understood that to allow the indirect fluorescence detection
part of the invention to operate, a fluorescent ion or fluorophore is
added to the buffer solution to become the principal component of the
electrophoretic buffer. Thus, when the ionic components of the sample
enter detector 20, laser-induced fluorescence results in either
displacement or ion-pairing of the ionic analyte with the fluorophore.
This produces a signal, from which can be derived the analytical results.
In FIG. 1, it can be seen that an analog signal from detector 20 can be
communicated to an analog-to-digital (A/D) converter 22, which interfaces
with a computer 24. Thus, the signals can be recorded, analyzed, and
manipulated to derive information about the separated and detected
constituent chemical components of the sample. It is to be understood,
that alternatively, detector 20 could be directly connected to an analog
chart recorder as an alternative method of recording the analytical
procedure.
It is also to be understood that in FIG. 1, a large resistive load 26 is
connected to the electrical line between power section 12 and capillary
section 14, and is directed to ground. The preferred embodiment resistive
load 26 comprises a 50 megohm (M Ohm), 100 watt resistor array to provide
a current shunt to ground by serving as parallel resistance to capillary
section 14.
Additionally, an interlock timer component 28 is utilized with power
section 12 to protect personnel from the high voltage when utilizing the
system, and to coordinate timed injection of the sample and the
electrophoresis procedure.
Finally, an ammeter 30 measures current continuously between what will be
referred to as the cathode end of capillary section 14 and ground.
FIG. 2 specifically and diagrammatically depicts the structure of detector
20. Capillary tube 18 passes through enclosure 32 of detector 20. A laser
34 producing a laser beam 36 positioned to direct laser beam 36 to
capillary tube 18 at a specific location. Laser beam 36 is first passed
through power stabilizer 38 and then into focusing lens 40, allowing laser
beam 36 to be precisely and narrowly focused in on a small location on
capillary tube 18. The angle between the laser and the capillary tube is
at Brewster's angle.
Laser beam 36 would then pass through capillary tube 18 but would induce
fluorescence changes in the elute which is passing by in capillary tube
18. These fluorescence changes are detected and monitored by PMT detecter
42 which is a photo-multiplier tube such as is known in the art. As shown
in FIG. 2, PMT 42 is positioned at an angle from the reflectance of laser
beam 36 from capillary tube 18. The fluorescence is imaged onto PMT 42 by
a ten power (10x) microscope objective 44. Stray light is minimized by
utilizing a spatial filter 46 in front of PMT 42. Finally, an interference
filter 48 is positioned directly in front of PMT 42 to isolate certain
fluorescence, as desired.
Thus, it can be seen that PMT 42 can be directly connected to A/D converter
22 and computer 24 as shown in FIG. 1 to record detection readings of the
fluorescence as a function of time.
In the preferred embodiment shown in FIGS. 1 and 2, the preferred
components are as follows:
Power section 12 is comprised of power source 50 which can be conventional
residential alternating current (102-130 VAC). High voltage power supply
52 is a 50 kilovolt (kV) Spellman model UHR 50PN150.
The interlock timer component 28 is circuitry which, as is known in the
art, automatically disconnects high voltage power source 52 if plexiglass
box 54, containing the anodic, high voltage end 56 from high voltage power
source 52 is handled. This serves as a safety feature for those using the
system. Interlock timer component 28 also contains an electronic timer
which is used to control the time of sample injection when samples are
introduced by electromigration of the sample solution into capillary tube
18.
Capillary tube 18 in the preferred embodiment is an untreated or silyated
100 centimeter (cm) fused silica capillary. It can be either 50
micrometers inside diameter (i.d.), available from SGE; or 15 micrometers
i.d., 150 micrometers outside diameter (o.d.), available from Polymicro
Technologies, Phoenix, Ariz. One end of capillary tube 18 is immersed in
buffer solution 16 in container 58 within plexiglass box 54. This serves
as the "anode" end of capillary tube 18. The other end of capillary tube
18 is immersed into buffer solution 16 in container 60, comprising the
"cathode" end of capillary tube 18.
Laser 34 comprises an HeCd (Helium Cadium) laser of 325 nanometer (nm)
wavelength at 8 milliwatts (mW), available from Liconix under product
model number 4240. Power stabilizer 38 stabilizes laser beam 36 to within
0.05% by utilizing laser power stabilizer model LS100, available from
Cambridge Research and Instrumentation, Cambridge, Massachusetts. The
stabilized laser beam at 5 mW is focused onto a small spot on capillary
tube 18 (in the preferred embodiment a 15 micrometer spot) with a 1
centimeter (cm) quartz lens 40 available from Melles Griot. In the
preferred embodiment utilizing capillary tube 18 with 15 micrometer i.d.,
and cleared of any polyamide coating, final detection volume is
approximately 3 picoliters (pL).
It is to be understood that capillary tube 18 is positioned at Brewster's
angle with respect to laser beam 36 at a position near the cathodic end of
capillary tube 18 (in the preferred embodiment 10 centimeters from the
cathodic end).
The ten power microscope objective 44 and spatial filter 46 are available
from commercial vendors, and are well known within the art. PMT 42 is
available from Hamamatsu under product designation R928. Interference
filter 48 in the preferred embodiment is at 405.1 nm, such as is
commercially available as is known in the art, or can be a broad band
glass filter available from Corning under product number 2-69, Corning,
N.Y.
In the preferred embodiment A/D converter is a five hertz (Hz) converter
available from Data Translation under model DT 2827, and computer 24 is an
IBM PC/AT.
Appropriate software, such as is well within the skill of those skilled in
the art, is utilized with computer 24.
It can therefore be seen that the present invention is operable to be used
as a reliable, efficient, and non-complex method of obtaining high
sensitivity and efficiency in the detection of chemical components of
substances. It does so nondestructively, without the requirement of
derivatization, by the indirect fluorescence detection.
The invention is universal in the sense that it is useful for many
different types of substances, including non-fluorescing compounds and
very small molecular compounds. It is fast in its resolution, and taking
only a few minutes for the complete process. It can be used on samples of
less than one picomole, and for sample volumes less than two nanoliters
(nL). Its detection limit is around fifty attomoles (amol). For
non-fluorescing samples, the system can "visualize" the chemical makeup by
utilizing the fluorescing species in the eluent.
Application of the invention is particularly useful as to biotechnical and
biochemical substances. Examples are nucleic acids and amino acids in DNA
sequencing or protein sequencing. Such things as peptides, nucleotides,
fatty acids, sugars, and glycolytic intermediates can be detected in their
native states, which is generally difficult if not impossible with
conventional methods. The procedure of the invention is thus useful in the
genetics field, studying metabolism, and even having direct analysis of
cells in vivo in clinical applications. Viruses and bacteria can be
studied as well as other difficult to detect and analyze substances. The
invention is even applicable to studying the chemical composition of
single cells, which could have tremendous effect on such things as
studying mutagens in cell cultures. The applications and potential
advantages are virtually innumerable.
It is believed to be further helpful to describe operation of the invention
with regard to the preferred embodiment depicted in FIGS. 1 and 2. Certain
enhancements and features will be pointed out. The buffer solution 16 is
prepared by choosing a fluorescing anion as part of the buffer solution
16. Containers 58 and 60 are then filled with buffer solution 16. A sample
is then dissolved into buffer solution 16 in container 58 and the system
is ready to begin.
It is to be understood that to increase the efficiency of the system, prior
to use, it is preferred that certain standards are maintained and
procedures followed. All chemicals used should be reagent grade unless
otherwise noted. All water used should be deionized, such as that
available from Millipore, Bedford, Mass.
Pre-preparation of capillary tube 18 consists of silating with
trimethoxychlorosilane (alternatively referred to as TCMS, available from
Aldrich). This is accomplished by aspirating a 20% solution of TCMS in
methylene chloride through capillary tube or column 18. The solvent is
evaporated, tube 18 ends are sealed, and tube 18 is then heated at
340-350.degree. C. for one to two hours. Tube 18 is then washed
successively with methanol, distilled water, and buffer solution 16 prior
to use.
The purpose of silating capillary tube 18 is to minimize interactions
between the ions in any injected sample on the surface of capillary tube
18, and thus reduce electroosmotic flow. This in turn plays a significant
part in reducing background noise in the indirect fluorescing signal. Both
peak-broadening and long-term drift of the separation process are
significantly reduced by the deactivation of the capillary tube 18's
surface. This in turn allows the use of more dilute buffer solutions (for
example, 50 micromoles of salicylate) and provides a much more stable
background fluorescence.
The sample is introduced to capillary tube 18 by electromigration from the
container 58. This can be accomplished by presenting electrical power
through the system for a regulated time period. Resistive load 26 is
advantageous in assuring standardization of injections using the
electromigration procedure. The amount of sample introduced by this method
is dependent on the rate at which capillary tube 18 is charged and
discharged. The rise time to maximum voltage is determined by the time
constant of high voltage power supply 52. The discharge rate on the other
hand is determined by the resistance of load 26. It is to be understood
that when only column or tube 18 is present, the fall time is determined
by the time constant of the discharge across tube 18, which varies with
tube dimensions and buffer concentration. When the resistance of load 26
is much lower than that of tube 18, the discharge rate will be dominated
by the time constant determined by load 26. Therefore, use of load 26 will
produce shorter, more uniform injections.
By interlocking the power supply to plexiglass box 54, which isolates the
anodic high voltage end 56 of the power supply, safety for the operator is
obtained. In other words, any handling of container 58, capillary tube 18
(at least at its anodic high voltage end 56), will disable electrical
power to plexiglass box 54.
Once a sample is injected into tube 18, detector 20 is powered, and A/D
converter 22 and 24 are made ready. Another feature to enhance the
reliability and efficiency of the system is to stabilize the output power
of laser 34. This stabilization is accomplished by feedback control from
power stabilizer 38 which determines the stability of background
fluorescence, which in turn are dependent on the stability of the light
source and the intensity of fluorescence. Although power stabilizer 38
does reduce laser power significantly using short focal-length focusing
lens 40, it is possible to focus to a spot less than 15 micrometers in
diameter on tube 18.
Stabilization of laser 34 increases the dynamic reserve by over 10.sup.3.
Because this allows reliable detection of very small detection volumes in
tube 18, this results in very high mass sensitivity for detector 20.
Concurrently, improved dynamic reserve also gives significant improvement
in separation efficiency. The increase in dynamic reserve allows use of
more dilute samples, decreasing sample loading, since the major ionic
component in the electrophoretic buffer solution 16 must be the floraphore
used to provide the signal.
The system is then operated and fluorescence is collected perpendicular to
the plane containing laser beam 36. In one preferred embodiment and method
of operation described here, salicylate fluorescence is isolated by
interference filter 48 at 405.1 nm. As a sample electromigrates down tube
18, this fluorescence is monitored. Until the sample reaches detector 20,
a constant background signal is detected. When the sample reaches detector
20, the interaction with the fluorophor will result in either displacement
(for ions of like charge) or ion-pairing (for oppositely charged ions)
with the fluorophor. The signal produced is thus independent of the
spectral properties of the analyte molecule and combines the innate
sensitivity of the fluorescence technique with a much broader spectrum of
analysis.
Either a chart recorder or computer 24 monitors the signal and makes a
record of it for derivation of ultimate results. It is to be understood
that computer 24 can also be programmed to perform digital data operations
according to desire.
The included preferred embodiment is given by way of example only, and not
by way of limitation to the invention, which is solely described by the
claims herein. Variations obvious to one skilled in the art will be
included within the invention defined by the claims. It can therefore be
seen the invention achieves at least all of its stated objectives.
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