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Description  |
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FIELD OF THE INVENTION
The present invention relates to the field of detecting analytes in an
analytical sample using a capillary separation system. More particularly,
the present invention relates to apparatus and methods for the detection
of analytes in samples using a capillary separation system incorporating a
noise suppression system.
BACKGROUND OF THE INVENTION
Capillary electrophoresis (CE) is one of the most powerful separation
methods. Analysis can be conducted even when samples are sub-nanoliter in
volume (Ewing, A. G.; Wallingford, R. A.; Olefirowicz, T. M., Anal. Chem.,
61, 292A-303A, 1989).
In capillary electrophoresis, a capillary containing buffer is suspended
between two reservoirs filled with buffer. An electric field is applied
across the two ends of the capillary. Analytes in a sample introduced at
the high potential end migrate toward the low potential end under the
influence of the electric field. Typically, capillary electrophoresis is
carried out with a 0-60 KV DC power supply. When an analyte is about to
exit the capillary, it can be detected by various techniques.
Commonly used methods for detecting analytes in CE include absorption,
fluorescence, electrochemical, and mass spectrometric detection (Ewing et
al. supra). Among these methods, UV-vis absorption detectors are the most
popular because of their versatility and simplicity, and because they are
usually supplied with commercial CE systems.
However, on-column absorption schemes in CE can only detect about 10.sup.-5
M to 10.sup.-6 M of an analyte in a sample due to the limited pathlength,
low intensity, and the presence of stray light when using an incoherent
light source (Bruin, G. J. M.; Stegeman, G.; Van Asten, A. C.; Xu, X.;
Kxaak, J. C.; Poppe, H. J., Chromatogr., 559, 163-181, 1991).
One way to improve the detection limit in CE is to increase the effective
pathlength. Taylor and Yeung increased the effective pathlength of their
absorbance detectors by directing the light beam along the capillary axis
to obtain an approximately 60-fold improvement in the pathlength (Taylor,
J. A.; Yeung, E. S., J. Chromatogr., 550, 831-837, 1991). The reduced zone
lengths associated with CE unit axial beam detection and the choice of
electrolytic buffers is limited to those with a refractive index higher
than that of the column walls.
Liquid chromatography (LC) is also a powerful analytical technique. LC
separates analytes in a mixture based on the repetitive distribution of
the molecules of the analytes between a mobile and a stationary phase. The
mobile phase is a liquid through which the analytes pass. In
high-performance liquid chromatography (HPLC), the driving force for the
movement of liquid and analytes is primarily the pressure difference
between the two ends of the chromatographic column. Mho and Yeung
disclosed a detection method for ion chromatography based on double-beam
Laser-Excited Indirect Fluorometry (Mho, S.; Yeung, E. S.; Anal. Chem.,
57(12), 2253-2256, 1985). Detection methods based on absorption for
analytes in LC are similar to those used in CE. As with any absorption
measurement, noise reduction will lead to increased sensitivity.
Lasers are not widely used in conjunction with CE or LC because of their
instability which increases noise, thereby hindering the detection of low
analyte concentrations in small samples. One way of reducing noise is to
use a dual beam detector system. In a conventional double-beam absorption
detector, the light output is split into a sample and a reference beam.
The resulting photocurrents, or voltages, are either subtracted from each
other or divided. Subtraction requires extremely fine adjustment of the
two beams to equal intensities and requires identical detector and
amplifier characteristics for good noise suppression. An analog divider is
typically used for conventional division and suffers from the poor
performance.
Hobbs, et al. describe a double-beam laser absorption system based on
all-electronic noise suppression (Hobbs, P. C. D., SPIE Proc., Roy, R.,
Ed., 1376, 216-221, 1991; Haller, K. L. and Hobbs, P. C. D., SPIE Proc.,
Feary, B. L., Ed., 1435, 298-309, 1991; Hobbs, P. C. D., Optics &
Photonics News, 17-23, April 1991). The system functions by subtracting
the signal from the reference photocurrents under feedback control to
cancel spurious modulation of the laser beam and excess noise, which is
the noise above the shot-noise level. Multiple spectral scans of a sample
in a cell containing I.sub.2 were subjected to analog low frequency (100
Hz high pass) filtering and signal-averaging (1000 individual sweeps) to
achieve a noise-equivalent absorption (i.e., noise in absorption) of
2.times.10.sup.-7 (Haller, K. L. and Hobbs, P. C. D., SPIE Proc., Feary,
B. L., Ed., 1435, 298-309, 1991).
Hobbs (U.S. Pat. No. 5,134,276) discloses electrical circuits for noise
suppression in such a system. The systems described by Hobbs et al. in the
aforementioned documents were, however, applied in spectroscopy and are
not related to CE or LC, which are low frequency operations.
To achieve noise reduction, Hobbs et al. scanned the output repetitively
and averaged the signals. While that process may improve the output
accuracy, it prevents the system from providing "real-time" or "on-column"
data of the changing levels of analyte in the sample. Furthermore, in
technologies such as biotechnology, analyses often have to be conducted on
samples which are small in volume and contain very dilute analytes. By
requiring multiple scans, such systems may not be able to accurately
analyze small, dilute samples.
As a result, there is a need for a capillary separation system which is
capable of providing highly sensitive, real-time detection of analytes
which may be dilute and/or provided in small samples.
SUMMARY OF THE INVENTION
The present invention provides a noise-suppressing capillary separation
system (NSCSS) for detecting the real-time presence or concentration of an
analyte in an analytical sample.
The NSCSS comprises a capillary separation means, such as CE or LC for
moving the analyte through a capillary, a source of coherent light for
providing a coherent light beam that is split into a reference beam and a
sample beam which irradiates and is transmitted through the capillary, and
a detector for detecting the reference beam and the transmitted light
through the capillary.
The invention also includes a noise suppressing electrical circuit for
noise suppression by electronically subtracting current derived from the
detected reference beam from current derived from the light transmitted
through the capillary. As used herein, the term "subtracting" refers to
finding the difference between two values.
Further, the present invention is also directed to a method of improving
the real-time detection of the presence or concentration of an analyte in
a sample analyzed in a capillary separation means. In this method, a
coherent light beam is split into a reference beam and a sample beam to
irradiate the sample in the capillary. The reference beam and the
transmitted light through the capillary are detected with a detector
having a noise suppressing electrical circuit.
The NSCSS according to the present invention provides a number of
advantages over known noise suppression systems for capillary separation
systems.
The analyte can be detected by the present invention in "real time" and "on
column" as the analyte passes through the capillary, i.e. any change of
concentration of the analyte as it passes through the detection zone of
the capillary can be detected essentially immediately without having to
sample from the capillary for later analysis or signal average to reduce
noise.
There is no need to carefully match electronic components used in the
systems according to the present invention. The only requirements are a
matched differential pair, a large collector coefficient B, and operation
in the active region. As long as each of the photodiodes is in the linear
response range, the photodiodes need not even be matched.
There is also no need for careful tuning of the electronics because the
negative feedback can be applied to keep the net DC photocurrent at zero
to result in noise cancellation and shot-noise limited performance.
The NSCSS of the present invention is particularly well-suited for noise
suppression in a low frequency analytical method such as CE or LC.
Typically, the elution of analytes in the capillary is slow enough that
data points do not have to be collected more frequently than once every
second. Using the NSCSS of the present invention, significant noise
suppression can be achieved without multiple scans and signal-averaging,
thereby increasing the response of the system for better real-time data.
Noise can typically be suppressed to 10 times the shot-noise-limited level
without signal-averaging when using the NSCSS and methods of the present
invention. If desired, however, signal-averaging can be performed
electronically or manually by averaging data from multiple measurements in
the application of the present invention, such signal-averaging is not
necessary to realize the noise suppression benefits of the invention.
The present invention can be used to measure analytes such as inorganic
ions and organic ions. The presence or concentration of biochemical
substances, such as polypeptides, polynucleotides, carbohydrates, cells,
bacteria, and viruses can be determined by utilizing the present
invention. Derivatives can be made from these substances for separation
using CE or LC. Using the direct absorption CE method of the present
invention, an analyte can be detected at a concentration that is about 10
to about 100 times the analyte concentration that is 8 shot-noise-limited,
typically in the range of about 10.sup.-8 M to about 10.sup.-7 M.
Direct absorption of laser light is generally thought to be inappropriate
in CE because lasers are typically highly unstable (.+-.0.1% in ideal
cases), degrading the dynamic reserve of the system, thus causing
difficulties in detection. However, CE, particularly indirect absorption
CE, is useful for detecting analytes in small samples. For example, in the
analysis of single cells, there is often a limited supply of sample.
The increased sensitivity of NSCSS according to the present invention can
be advantageously utilized to detect small samples in a capillary with a
small i.d. Using the present invention in indirect absorption in CE, an
analyte can be detected at a concentration that is about 20 to about 200
times the analyte concentration that is shot-noise-limited, typically in
the range of about 10.sup.-7 M to about 10.sup.-6 M. Likewise, the present
invention increases the sensitivity of detecting an analyte using LC when
compared to conventional LC detection systems.
The detection limit is inversely proportional to the capillary inside
diameter. Even with capillaries as small as about 20 .mu.m in inside
diameter, using a laser beam diameter of about 10 .mu.m, there is little
alignment noise. Comparatively, the sensitivity in commercially available
detectors deteriorates much faster with the decrease of capillary
diameter. With commercial systems, a capillary smaller than 50 .mu.m i.d.
does not usually provide good sensitivity.
In systems according to the present invention, the collimated laser beam
can easily be focused down to a spot of a few micrometers. In this way,
almost all of the light passes through the inside core of the capillary
along a diameter and Beer's Law will hold, resulting in low noise compared
to commercial systems. This is true for capillaries in the NSCSS of as
small as 20 .mu.m i.d. or less.
For a better understanding of the features and advantages according to the
present invention, reference should be made to the following description
of the preferred embodiments and methods.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of an embodiment of an on-column noise
suppressing capillary separation system.
FIG. 2 is a schematic diagram for an embodiment of a noise suppressing
electrical circuit.
FIG. 3 is a graph of log (log-out voltage) vs. log (malachite green
concentration) in an embodiment using NSCSS of the present invention.
FIG. 4 is a graph of the absorbance data obtained in the analysis of
5.times.10.sup.-7 malachite green in a commercial CE system.
FIG. 5 is a graph of the log-out voltage data obtained in the analysis of
2.times.10.sup.-8 malachite green in a NSCSS of the present invention.
FIG. 6 is a graph showing the linear-out voltage data obtained in the
analysis of 4.times.10.sup.-7 malachite green in a NSCSS of the present
invention.
FIG. 7 is a graph of the log-out voltage versus time data of indirect
absorption detection by analysis of 1.5.times.10.sup.-7 M pyruvic acid
sodium salt in CE with a capillary of 75 .mu.m i.d. using a NSCSS of the
present invention.
FIG. 8 is a graph of the transmitted light intensity versus time data of
indirect absorption detection in the analysis of 1.5.times.10.sup.-6 M
pyruvic acid sodium salt in CE with a capillary of 75 .mu.m i.d. using a
commercial system.
FIG. 9 is a graph of the log-out voltage versus time data of indirect
absorption detection in the analysis of 6.times.10.sup.-6 M pyruvic acid
sodium salt in CE with a capillary of 14 .mu.m i.d. using a NSCSS of the
present invention.
FIG. 10 is a graph of the transmitted light intensity versus time data of
indirect absorption detection in the analysis of 3.times.10.sup.-4 M
pyruvic acid sodium salt in CE with a capillary of 14 .mu.m i.d. using a
commercial system.
FIG. 11 is a graph of the log-out voltage versus time data of indirect
absorption detection by analysis of 1.5.times.10.sup.-5 M pyruvic acid
sodium salt and 1.5.times.10.sup.-5 M NPAS in CE using a NSCSS of the
present invention.
FIG. 12 is a graph of the log-out voltage versus time data of cation
indirect absorption detection in the analysis of 4.times.10.sup.-6 M
potassium nitrate in CE using a NSCSS of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is a noise-suppressing capillary separation system
(NSCSS) for detecting the real-time presence and/or concentration of an
analyte in a sample. The present invention is also directed to a method of
using the noise-suppressing capillary separation system to provide a
real-time indication of the presence and/or concentration of an analyte in
a sample. The NSCSS contains a capillary separation means such as a CE or
LC system for the movement and separation of analytes. The presence and/or
concentration of the analyte is detected by a detector at a detection zone
near the exit end of the capillary.
The analyte can be detected by the present invention in "real time" and "on
column" as the analyte passes through the capillary, i.e. any change of
concentration of the analyte as it passes through the detection zone of
the capillary can be detected essentially immediately without having to
sample from the capillary for later analysis or signal average to reduce
noise.
Laser energy is preferably used to provide a coherent light beam that is
split into two: one as a reference beam and one as a sample beam for
irradiating the analyte in the detection zone of the capillary. The
absorbance of the analyte in the detection zone of the capillary is
measured by a detector which has a noise suppressing electrical circuit.
I. Capillary Electrophoresis and Liquid Chromatography
As applied in the present invention, capillary electrophoresis system or
liquid chromatography system that contains a capillary effective for use
with a laser of suitable wavelength for detecting the analytes can be
used. The present invention can be used with open-tube capillary
electrophoresis as well as for capillary electrophoresis involving
capillaries that contain separation-medium. Open tube capillary
electrophoresis is useful in analyzing samples containing substances that
have a tendency to plug a capillary with solid phase medium, for example,
samples of cells, bacteria, or viruses. This invention is likewise
suitable for use in liquid chromatography for noise suppression and
improving detection limits. Capillary electrophoresis systems and liquid
chromatography systems are well known in the art and are commercially
available. As used herein, the term "capillary separation means" refers to
a system that is either capillary electrophoresis (CE) or liquid
chromatography (LC) wherein a capillary is used for separating analytes in
a sample. Because the LC column in analytical LC is usually small in
diameter, as used herein, the term "capillary" refers to a capillary in CE
or a column in LC. Typically, such a capillary has an inside diameter of
about 2 .mu.m to 200 .mu.m, more often about 14 .mu.m to 100 .mu.m.
In CE the capillary is preferably made of fused silica because fused silica
is superior to other materials such as polymers and metals in permitting
transmittance of coherent light of preferred wavelength in the UV region.
In LC, even if the capillary is made of a nonsilica material, preferably
at least the detection zone is made of fused silica or other materials
which transmit UV radiation.
Interactions such as absorption of chromophores on the capillary wall can
introduce noise. In cases wherein one chromophore is positively charged,
the positively charged chromophore and negatively charged capillary wall
may interact much more strongly than in the case of indirect anion
detection. Therefore, coated capillaries are more favorable for indirect
cation detection, especially when large molecules are used as
chromophores. Coatings are commonly made with hydrophobic polymers. An
example of an effective coating method is silation. Silation also reduces
peak broadening of the eluting sample and long term drift. Methods of
silating a fused silica capillary are well known in the art.
Typically, the outside surface of a fused silica capillary in CE is coated
with a polyimide or polyamide. Near the exit end of the capillary, a
detection zone can be created for the transmission of light by removing
the polyimide or polyamide coating. The removal of the polyimide or
polyamide coating can be accomplished by dipping the end of the polyimide
or polyamide-coated fused silica capillary in concentrated sulfuric acid.
A. Direct Absorption Detection
An analyte containing a chromophore can be analyzed by eluting through a
capillary in the CE or LC system. Before the chromophores reach the
detection zone of the capillary, the transmitted light detected at the
detection zone represents the background signal. As the chromophores pass
by the detection zone, part of the irradiated laser radiation is absorbed
by the chromophores. The transmitted light can be detected by the detector
to determine the absorbance of the chromophores. The absorbance can be
analyzed to obtain information on the presence and/or concentration of the
chromophore, and therefore, of the analyte in the fluid passing through
the detection zone. Representative suitable chromophores are color dyes
such as malachite green which absorbs light of 633 nm wavelength. Other
chromophores for absorbing light of other wavelengths are also known in
the art.
The chromophores can be the analyte, or more commonly, the analytes are
labeled, or tagged with a suitable chromophore before being injected into
the capillary. Such labeling and tagging techniques are well-known in the
art.
B. Indirect Absorption Detection
An analyte can also be detected without directly labeling or tagging before
being injected into the capillary. A buffer with a chromophore can be
passed through the capillary. When an analyte is injected into the
capillary and reaches the detection zone, the interaction of the
chromophore being transferred to (i.e. ion-pairing with) or being
displaced by the analyte, thereby leading to a change in the absorbance of
the fluid in the detection zone. The change in absorbance can be detected
to determine the presence or concentration of the analyte.
For indirect absorption detection of anions, bromocresol green can be
selected as the chromophore. Bromocresol green is soluble in water, very
stable, and has a large molar absorptivity (.epsilon.=7.times.10.sup.4). A
pH of about 8.8 it is almost completely disassociated. Bromocresol green
also has little absorptive interaction with the capillary wall. Thus, this
compound is suitable as a chromophore for indirect detection.
C. Collimation of Coherent Light in Capillary
In the analysis of the concentration of an analyte by absorbance
measurement, the relation between the absorbance, the concentration of the
analyte solution, and the light pathlength of the measuring device can be
mathematically described. Preferably, the laser beam is directed to
irradiate the capillary substantially orthogonally so that the beam passes
through the diameter (i.e., the center) of the capillary.
However, in a capillary, if the diameter of the laser beam is not
adequately small compared with the capillary inside diameter, the laser
beam will pass through the solution inside the capillary with a
distribution of pathlengths. The pathlength of the light in the beam can
vary greatly depending on the position of the light in relation to the
capillary. Such variations contribute to noise in the measurement of the
absorbance of the analyte in the column.
As a result, the effective optical pathlength is much smaller than the
inside diameter (i.d.) of the capillary. Further, part of the beam may be
refracted by the wall of the capillary and is not detected by the
detector. Lower light intensities due to poor collimation will lead to a
lower signal-to-noise ratio. To reduce noise, it is therefore preferable
to collimate substantially the whole light beam to irradiate through the
axial center of the capillary. Generally, the laser beam directed to the
capillary is collimated into the capillary with a beam diameter of less
than about 0.25 of the insider diameter of the capillary. Preferably beam
diameter is about 0.02 to about 0.25 times the insider diameter of the
capillary.
It should be noted that although a He-Ne laser producing a coherent light
of 632.8 nm was used in the preferred embodiment, such a laser is not
essential and can be replaced by any source of coherent light with good
pointing stability and suitable wavelength. By using UV lasers, one will
be able to detect different kinds of analytes, especially proteins,
nucleic acids and their constituents. Following the teaching of this
invention, the selection of a laser of suitable wavelength and power can
be accomplished following routine practices.
II. Noise Suppression Detector
Shot noise is the result of random current fluctuation of a statistical
nature. When a photodetector is used for detecting light, the number of
photons striking the photodetector in a specific time interval has a
Poisson distribution, which leads to a variation of the current generated
by the photodetector. A detector system that has eliminated all other
noise except shot-noise is referred to herein as "shot-noise-limited."
Noise and spurious modulation in light can be suppressed or substantially
eliminated from the data generated by a detector using photodetectors if a
laser beam is split into a sample beam and a reference beam and the
photocurrents generated by the two beams electronically subtracted one
from the other.
In the preferred noise suppressing electrical circuit used in the present
invention, a sample photodetector, preferably a photodiode, is used to
generate a sample current when struck by light from the sample beam which
is transmitted through the capillary. The sample current represents the
composite of an informational signal impressed upon a carrier current. The
carrier current represents a sample steady state signal modulated by
noise. The sample steady state signal (as modulated by the noise) is
represented by a sample steady state current.
A reference photodetector, preferably a photodiode, generates a reference
current when struck by a reference beam. The reference current represents
a reference steady state signal modulated by noise.
Current dividing means is provided for dividing the reference current into
first and second components. The first component of the divided reference
current includes a divided steady state signal which is preferably
approximately equal in amplitude to the sample steady state signal of the
sample current.
As a result, the first component of the divided current and the sample
current are combined to reduce or, preferably, substantially cancel, the
sample steady state signal of the sample current, thereby producing an
output current indicative primarily of the informational signal of the
sample current. In other words, one current (typically the first component
of the divided reference current) is subtracted from the sample current to
cancel out the sample steady state signal, thereby producing an output
current which is free of what can be referred to as an output steady state
signal. In those instances where the steady state signals are not
cancelled out, the output current may contain an output steady state
signal, i.e., the portion of the sample steady state signal not cancelled
by the first component.
Although it is preferable that the reference current be larger than the
sample current, the noise suppressing electrical circuit can also be used
when the sample current is larger than the reference current. In that
case, the sample current is divided into two components to cancel the
sample steady state signal to obtain an output current which corresponds
to the informational signal in the same manner as described above.
A. Optical Arrangement
A schematic diagram of one preferred double-beam laser absorption detector
for CE is shown in FIG. 1. The light source is a 10 mW He-Ne laser 110
(GLG5261, NEC, Mountain Valley, Calif.), which operates at 632.8 nm. The
laser beam 114 is split into a sample beam 116 and a reference beam 118.
To split a laser beam this way, the laser beam 114 is reflected by a
mirror 120 to direct the laser beam 114 through a polarizer 126, which
polarizes the beam 114 into two separable components.
The polarized beam is then passed through a calcite beam displacer 130
(Karl Lambrecht Corp., Chicago, Ill.) to split the beam into orthogonally
polarized sample and reference beams 116 and 118.
The polarizer 126 eliminates polarization noise, which can cause
unsuppressible, uneven intensities in the calcite beam displacer 130. The
calcite beam displacer presents different refractive indexes for the
polarized light components, enabling them to travel in different paths.
The calcite beam displacer 130 is rotated so that the reference beam 118
is made to be about twice as intense as the sample beam. Although such a
method of splitting a laser beam is preferred, other means of splitting a
laser beam, such as a half mirror, can also be employed.
The reference beam 116 is directed through a 5 mm diameter aperture 134 and
strikes a photodiode 126, generating a reference photocurrent. The sample
beam 116 is directed to a lens 140 by reflecting off a second mirror 144
(Newport). Lens 140, a 1-cm focal length quartz lens (Melles Griot Corp.,
Irvine, Calif.) is used to focus the laser beam 116 into a detection zone
150 made by removing a 5-mm section of polyamide coating from the
fused-silica capillary 156 near the exit end of the capillary.
The inlet end of the capillary 156 is connected to a high voltage reservoir
146 and the exit end of the capillary 156 is connected to a grounded
reservoir 148. The capillary 156 is preferably mounted on a precision x-y
positioner (not shown) (Newport, 462 Series) for fine alignment of the
capillary 156 in relation to the laser beam 116.
On the side of the capillary 156 opposite the side irradiated by the laser
beam 116, light transmitted through the capillary detection zone 150 is
focused by a 35-cm focal length quartz lens 154 (Melles Griot Corp.) and
directed through a 5 mm diameter aperture 160. The laser beam 164 that
passes through the aperture 160 is monitored by the sample photodiode
(first photodiode) 170. The photocurrent generated at the reference
photodiode (second photodiode) 136 and the sample photodiode 170 are
directed to the noise suppressing electrical circuit 174, which is
electronically connected to a computer 180 for analyzing the electrical
data generated in the noise suppressing electrical circuit 174.
The apertures 134 and 160 isolate the sample beam 118 and the reference
beam 116 from each other to avoid crosstalk, i.e., the light from one beam
striking the photodiode intended for the other beam. The size and position
of the apertures are carefully chosen to avoid vignetting, which is the
result of part of the beam being blocked while the beam passes through the
aperture. Vignetting causes noise if the beam moves.
The exact aperture size is determined based on variables within each system
by simple experimentation. In one preferred method of determining aperture
size, the capillary is aligned in relation with the laser beam to avoid
Fabry-Perot fringes, which are interference patterns in the transmitted
light. Vibrations in the capillary can change the alignment and introduce
noise. Such vibration-caused noise can be substantially reduced by
affixing the capillary on a solid mount and the detection zone of the
capillary on an adjustable x-y positioner.
B. Noise Suppressing Electrical Circuit
One preferred noise suppression circuit used in the present invention is
similar to those described by Hobbs et al., supra. FIG. 2 shows the
schematic diagram of an embodiment of the noise suppression electronic
circuit of the present invention. Two 12 v batteries 210 and 212 in series
are used to drive the electrical circuit. Though power supplies can be
used for powering the electrical circuit, preferably batteries are used
because they tend to reduce noise in the circuit.
Two photodiodes 136 and 170 (BPW34, Siemens) serve as sample and reference
beam detectors. The reference photodiode (second photodiode) 136 generates
a reference steady state current when struck by the reference beam. The
sample photodiode (first photodiode) 170 generates a sample current when
struck by the transmitted light directed from the capillary detection
zone. The sample current represents an informational signal modulating a
steady state current. A PNP bipolar transistor Q.sub.3 (2N3906) is used to
prevent the capacitance of the sample photodiode 170 from loading the
summing junction 230 of the operational amplifier, A1.
A pair of bipolar transistors (Q1,Q2) (Motorola MAT04), in a differential
pair configuration, acts as a variable current divider. This current
divider is electronically controlled to divide the reference current
passed from reference beam photodiode 136 into two component currents. One
of the two component currents, referred to as the first component,
preferably has substantially the same steady state current amplitude as
the steady state current amplitude of the sample current generated by the
sample photodiode 170.
This first component current is subtracted from the sample current at the
summing junction 230 of the operational amplifier A1 so that the steady
state currents of the two substantially cancel out or at least reduce the
steady state signal portion of the sample current, resulting in an output
current that is substantially free of a component caused by excess noise,
i.e., the steady state current of the output current at the summing
junction is approximately zero. Preferably, the two currents have steady
state amplitudes that are within 0.1% of each other, although any
reductions in the steady state current portion of the output current are
desirable.
The operational amplifier A1 (Motorola OP-27) converts the resulting output
current at the summing junction 230 to a voltage. The operational
amplifier A2 integrates the output voltage of A1 and feeds back to the
differential pair Q1-Q2, which controls the division of the reference
photocurrent. In the embodiment, R.sub.F is 20 K.OMEGA., R.sub.1 is 1
K.OMEGA., R.sub.2 is 1 K.OMEGA., R.sub.3 is 24 .OMEGA., C is 2.2
microfarad. The circuit is shielded from the environment in the preferred
embodiment by a piece of aluminum foil. Optionally, a .+-.15 V DC power
supply (BK Precision, Model 1660, Chicago, Ill.) can be used to drive the
noise suppressing electrical circuit.
III. Output from Noise Suppressing Capillary Separation System
The preferred noise suppressing electrical circuit has both a linear
output, or linear-out (A1) and a log output, or log-out (A2).
A. Log Output
Log-out voltage, V.sub.log of A2 provides a low-pass filtered voltage
related logarithmically to the sample and reference beam intensities (see
Haller, K. L. and Hobbs, P. C. D. SPIE Proc., Feary, B. L., Ed., 1435,
298-309, 1991):
##EQU1##
Because the output from A2 is integrated, it typically contains less noise
than the linear output at A1. The normalized transmittance is then
##EQU2##
where I is the intensity of the transmitted light through the capillary
and I.sub.0 is the transmitted light in the absence of absorption. V is
the voltage registered from log-out in volts and V.sub.0 is the log-out
voltage in the absence of absorption. Since V.sub.0 is set to be about -70
mV and V is small in a low concentration sample, the exponentials can be
expanded to obtain:
##EQU3##
where .DELTA.V=V.sub.0 -V is the logout voltage change (i.e., peak
height). Rearranging the equation, we obtain:
##EQU4##
Based on Beer's Law, the transmittance is:
##EQU5##
where .epsilon. is the molar absorptivity of the sample, b is the
pathlength, c is the sample concentration, and k is a constant for a given
reference beam and sample beam ratio.
For a low concentration sample, I.sub.0 /I is about equal to 1 and thus,
##EQU6##
Consequently, a relationship between the concentration of a sample and
log-out voltage can be derived:
.DELTA.V=(2-V.sub.0) ln 10 .epsilon.bc=k .epsilon.bc
where k is equal to {(2-V.sub.0) ln 10}. This equation provides an
alternative for concentration calculations. A concentration versus peak
height calibration curve for malachite green in a capillary separation
system such as CE or LC can be constructed.
In the noise suppressing electrical circuit of the present invention as
described hereinabove, the RMS noise density of the log-out voltage is:
##EQU7##
where q is the electron charge. In the NSCSS of the present invention, one
can estimate the maximum log-out voltage within which the ratio of the
collector currents of the two transistors, Q.sub.1 and Q.sub.2, is
dependent on the difference voltage .DELTA.V.sub.BE =V.sub.BE2 -V.sub.BE1.
The ratio of the collector currents for the two transistors can be
expressed as: | | |
