Note: Descriptions are shown in the official language in which they were submitted.
~ 3~72 64680-433D
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.. This application is a divisional of copending Canadian
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:: patent application ~erial No. 558,183 filed on February 4, 1988.
This invention relates to a sensor system, particular-
ly to a system for determining the pH of a liquid medium and a
. system for determining the concentration of carbon dioxide in a
liquid medium. The invent.ion is also concerned with a method for
:,~ measuring the concentration of carbon dioxide in a medium.
~ The measurement of desired parameters in various
.~ media, particularly in biological systems, is frequently required.For example, the measurement in blood of pH levels and concen-
tration of gases, particularly oxygen and carbon dioxide, is
: important during surgical procedures, post-operatively, and
during hospitalization under intensive care and many devices for
the measurement of said physiological parameters have been
suggested in the art.
United States Patent No. 4,003,707, Lubbers et al, and
;:~ its reissue patent Re 31879, disclose a method and an arrangement
.~ for measuring the concentration of gases and the pH value of a
. sample, e.g. blood, involving the use of a fluorescent indicator
, 20 at the end of a light-conducting cable which is sealingly covered$~ by or embedded in a selectively permeable d.iffusion membrane.
;
The radiation transmitted to and emitted from the indicator must
:. be passed through various filtering elements and light elements,
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including reflectors~ beam splitters and amplifiers before any
meaningful measurements can be made.
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,~ United States Patent No. 4,041,932, Fostick, dis-
closes a method whereby blood constituents are monitored by
measuring the concentration of gases or fluids collected
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in an enclosed chamber sealingly attached to a skin
"window" formed by removing the stratum corneum over a
small area of the patient's skin. The measurements in
the enclosed chamber are made, inter alia, by determining
S the difference in intensity of light emitted from a
fluorescent indicator.
U.S. Patents No. 4,200,110 and 4,476,870, Peterson
et al, disclose the use of a pH sensitive indicator in
conjunction with a fiber optic pH probe. In each of
these patents the dye indicator is enclosed within a
selectively permeable membrane envelope.
U.S. Patent No. 4,548,907, Seitz et al, discloses
a fluorescent-based optical sensor comprising a mem-
brane immobilized fluorophor secured to one end of a
bifurcated fiber optic channel for exposure to the
; sample to be analyzed.
Many fluorescent indicators sensitive to pH, and
i thereby useful for PCO2 measurements, are known in the
art. Examples of useful fluorescent indicators are
,~ 20 disclosed in the above patents and also in "Practical
Fluorescence" by George E. Guilbault, (1973) pages
;~ 599-600.
Sensor devices using fluorescent indicators may be
;~ used for in vitro or in vivo determinations of com-
ponents in physiological media~ For in vitro deter-
~ minations the size of the device is normally of no
-~ consequence, but for in vivo use, the size of the
sensor may be extremely critical and there is an
increasing need in the art to miniaturize sensor
devices r particularly catheter-type devices, for the in
vivo determination of components in physiological
media, e.g. blood. However, diminution in size of the
components of such devices, particularly in the size of
;:~ the sensor itself, decreases the strength of the signal
'` 35 emitted by the indicator and consequently presents
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problems in the detection and measurement of said
signal. These problems are aggravated when the
detector system requires a multiplicity of components,
such as filters, beamsplitters and reflectors to
isolate and measure the emitted energy. Each of said
components reduces the emitted signal strength
resulting in a sequential loss of measurable signal.
Consequently, the more components present in the
system, the weaker the final signal strength.
One approach for obtaining a meaningful
measurement is to use the ratio of two signals which
provides a signal with greater resolution than that
;~ obtainable from prior art systems based upon a single
signal. Zhang ZHUJUN et al in Analytica Chimica Acta
160 (1984) 47-55 and 305-309 disclose that the
fluorescent compound 8-hydro~y-1,3,6-pyrenetrisulfonic
acid, referred to herein as HPTA, fluoresces when
-~ axcited by excitation radiation having wavelengths of
470 and 405 nm and the fluorescence emission is
sensitive to changes in pH in the physiological range
of 6 to 9.
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In contrast to the system disclosed by Zhujun et
al, which uses two excitation radiations to produce
fluorescence, surprisingly, it has now been found that
hiyhly accurate, stable determination of pH can be
obtained from a single external source of excitation
~`~ radiation which is used to excite a first fluorescent
~` indicator which in turn emits fluorescent radiation to
excite fluorescence emission in a second fluorescent
indicator, e.g. HPTA said first indicator being
. insensitive to pH.
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According to the present invention, a new improved
system is obtained by the use of two fluorescent
indicators acting in concert or by the use of a single
fluorescence indicator which emits fluorescent signals
of different wavelengths in different carriers which
signals have intensities proportional to the parameter
under investigation. Under this approach the parameter
being measured is determined by the ratio of two
diverging signals which provides greater resolution and
a highly accurate, stable determination.
The term "stable" as used herein is intended to
mean the stability of the determination with respect to
all factors which might influence the measurement other
than the parameter heing measured. Thus the determina-
tion is not affected by, for example, changes in the
strength of the excitation radiation, fluctuations in
light or temperature or minor equipment defects. Since
the quantity being measured is a ratio between two
given intensities and this ratio remains constant when
re 20 the value being measured is constant, irrespective of
th~ actual size of the individual intensities, the
'~ resultant determination is necessarily sta~le.
In accordance with the present invention there is
~` provided a sensor system for determining the p~ of a
'~ 25 liquid medium which comprises, in combination, a first
fluorescent indicator whose fluorescence emission is
insensitive to pH and a second fluorescent indicator
whose fluorescence emission is highly sensitive to
solution pH~ which indicator combination is adapted to
~` 30 respond when a source of excitation radiation of
r, wavelength ~ o is applied to the system such that said
first fluorescent indicator is selectively excited by
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said excitation radiation to emit pH-insensitive fluorescence
emission at wavelength ~1' which emission overlaps the excikat.ion
radiation spectrum of said second fluorescent indicator, and said
second indicator being excited by said emission radiation of
wavelength ~1 and in turn emitting a pH-dependent fluorescence
emission of wavelength ~2~ the ratlo of intensities of the
. radiation of wavelengths ~ 2 providing a highly accurate, stable
; determination of the pH of said liquid medium.
: The invention also provides a method for determining
the pH of a liquid medium which comprises contacting said medium
; with a sensor system comprising, in combination, a first fluo-
rescent indicator whose fluorescence emission is insensitive to
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. is highly sensitive to solution pHt sub~ecting said sensor system
~ to excitation radiation of a predetermined wavelength ~O, thereby
.~ selectively exciting said first fluorescent indicator to emit a
pH-insensitive fluorescence emission at a wavelength of ~1~ which
emission overlaps the excitation radiation spectrum of said
~:~ second fluorescent indicator and thus excites said second indi-
cator to emit a pH-dependent fluorescence emission of wavelength
'~ ~2' and measuring either ~i) the ratio of intensities of the
.~ emitted radiation of wavelengths ~ 2~ or (ii) the ratio derived
from the intensity of the emitted radiation of wavelength ~1 and
~ the intensity at the isobestic point between the emission curves
:~5 of said first and second indi~ations, thereby obtaining R hi~hly
accurate, stable determination of the pH of said liquid medium.
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,-~ The sensor system and method described above are
~ referred to h~rein as the first embodiment of the invention.
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By using the system and method of the invention,
enhancement of signal resolution is obtained due to the
divergence of the fluorescence emission intensities as
a function of pH of the surrounding medium. This
phenomenon provides a higher degree of measurement
resolution thus providing an increase in measurement
.. accuracy in determining solution pH.
: The invention further provides a sensor system for
the determination of the concentration of carbon
. 10 dioxide in a liquid medium which comprises, in combi-
. nation, a first fluorescent indi.cator whose fluore-
.(~ scence emission is insensitive to pH and a second
fluorescent indicator who.se fluorescence emission is
highly sensitive to solution pH, which indicator
. 15 combination is associated with a bicarbonate solution
.; bounded by a carbon dioxide-permeable membrane, and is
adapted to respond when a source of excitation
radiation of wavelength '~ o is applied to the system
:. such that said first fluorescent indicator is
~ 20 selectively excited by said excitation radiation to
`~ emit a pH-insensitive fluorescence emission at
wavelength ~ 1~ which emission overlaps the excitation
.. ;. radiation spectrum of said second fluorescent
indicator, said second indicator being excited by said
,~ 25 emission radiation of wavelength ~ 1' and in turn
emitting a pH-dependent fluorescence emission of
wavelength A 2~ the ratio of intensities of the
.`. radiation of wavelengths ,~ 2 providing an
$ii~ indication of the solution pH within the membrane and
.~ 30 thereby a highly accurate, stable determinatisn of the
~ concentration of carbon dioxide in the liquid medium.
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~ 1 326772
- 7 - 64680-433D
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. The invention still further provides a method for
determining the concentration of carbon dioxide in a liquid
:~ medium which comprises contacting said medium with a sensor
~ system comprising, in combination, a first fluorescent indicator
:
whose fluorescence emission is insensitive to pH and a second
'. fluorescent indicator whose fluorescence emission is highly sen
?
~ sitive to solution pH, which indicators are associated with a
;~ bicarbonate solution bounded by a carbon dioxide-permeable mem-
~ brane, subjecting said sensor system to excitation radiation of
.. ~ 10 a predetermined wavelength ~O, thereby selectively exciting said
. first fluorescent indicator to emit a pH-insensitive fluores-
:~ cence emission at a wavelength of ~1~ which emission overlaps
the excitation radiation spectrum of said second fluorescent
.i,
.~ indicator and thus excites said second indicator to emit a pH-
dependent fluorescent emission of wavelength ~2~ and measuring
either (i) the ratio of intensities of the emitted radiation of
~; wavelengths ~ 2~ or (ii) the ratio derived from the intensity
~; of the emitted radiation of wavelength ~1 and the intensity at
the isobestic point between the emission curves of said first
,.,~ .and second indications, thereby obtaining an indication of the
solution pH wlthin the membrane and thus a highly accurate,
stable determination of the concentration of carbon dioxide in
`~t, '
the liquid medium.
The sensor system and method for pCG2 determination
. described above are referred to herein as the second embodiment
~x of the invention.
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This second embodiment~ as with the first embodi-
'.~ ment, provides enhancement of signal resolution due to
`~: divergence of fluorescence emission intensities.
The invention yet further provides a method ofmeasuring the concentration of carbon dioxide in a medium by
determining the water content in a pH-
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8 646~0-~33
independent sensor system comprising an optical fiber having a
~ proximal end and a distal end, said distal end having attached
;~. thereto a fluorescence indicator embedded in a carrier matrix with
a predetermined water content, said carrier matrix containing a
~; miscible mixture of water and non-aqueous solvent in predetermined
. proportions and being separated from said medium by a gas-
permeable, water-impermeable diffusion membrane, said indicator,
....
. when excited by excitation radiation of a predetermined wavelcngth
O~ emitting fluorescent emission at a wavelength ~w in the
presence of water and at a wavelength ~s in the presence of said
non-aqueous solvent, the intensity of each emission being
. independent upon the ratio of water to non-aqueous solvent present
~, in th~ system such that the ratio of the intensities of emittcd
`;~ radiation of wavelengths ~w and ~s is therefore proportional to
~;: the amount of water present and diffusion of carbon dioxide
through said gas-permeable membrane and subsequent reaction with
~: water to deplete the water content of the system induces a change
; in the intensities of said emissionsj which method comprises
transmitting through said optical fiber, from a source adjacent to
its proximal end, excitation radiation of said predetermined
~: wavslenyth ~O, and measuring the ratio of the intensities of the
emitted radiation of wavelengths ~w and ~s~ thereby obtaining a
~,~ determination of the water content and calculating tllerefrom thc
carbon dioxide concentration in the surrounding medium.
The above-dsscribed method of measuring PCO2 as a
`.~ function of the water content in a pH-independent sensor system
~, and the system used in such method is
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~ 9 132~772
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referred to herein as the third embodiment of the
-; invention.
-, Here again, enhancement of signal resolution is
r, obtained from divergence of fluorescence emission
intensities.
The sensor system of the first embodiment of the
invention preferably includes an optical fiber having a
distal end and a proximal end, in which said
` combination of fluorescent indicators is attached to
said distal end and said proximal end is adapted to
receive excitation radiation from said source of
; excitation radiation.
The first fluorescent indicator, which is insensi-
tive to pH, is preferably 6,7~dimethoxycoumarin or a
pH-insensitive coumarin derivative. A typical
coumarin derivative is beta-methylumbelliferone,
particularly in the form where it chemically bonded to
an acrylic polymer. The pH sensitivity of the
umbellilferone polymer may be retarded by reacting the
~- 20 polymer solution with an excess of cross-linking agent
`~ such as poly (acrylic acid).
The particularly preferred indicator for the
purpose of the present in~ention is 6,7 dimethoxy-
coumarin which, when excited by excitation radiation
~'~ 25 having a wavelength of 337 nm emits fluorescent
radiation at a wavelength of 435 nm. The character-
`~ istic excitation and emission spectra of 6,7-dimethoxy-
-~: coumarin are illustrated in the accompanying drawings
as described hereinafter.
It is to be understood that when reference is made
herein to a particular wavelength, for example with
respect to excitation or emission, it is intended to
mean that wavelength which is most representative of
` the condition being described; most typically the peak
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of a curve illustrating the spectrum which fully
represents said condition. Thus, as shown by the curve
for the excitation spectrum, 6,7-dimethoxycoumarin is
excited by radiation over a spectrum of wavelengths
S from 310 to 380 with an optimum excitation at the peak
wavelength of 337 nm. For convenience, unless other-
wise defined, the wavelengths quoted herein are the
peak wavelengths for the phenomenon in question.
The preferred second indicator used in the first
embodiment of the invention is HPTA.
In the preferred first embodiment of the invention
excitation radiation having a wavelength, i.e. a peak
wavelength, ~ o, of 337 nm, for example from a
nitrogen gas laser, is transmitted from the proximal
lS end of an optical fiber through the distal end where it
excites a first indicator, pxeferably 6,7 dimethoxy-
coumarin, which emits fluorPscent radiation having a
wavelength, ~ of 435 nm. This fluorescen-e
emission, in turn, excites the second indicator,
preferably HPTA, to emit fluorescent radiation having a
wavelength, ~ 2~ of 510 nm.
The intensity of the fluorescence emission of
;r,' wavelength ~ 2 ~510 nm) is dependent upon the
,?, intensity of the excitation emission of wavelength A
and upon the pH of the surrounding liquid medium, so
that measurement of the ratio of the intensities of the
emitted radiation of wavelengths ~ 2 gives a
highly accurate, stable determination of the pH of said
, liquid medium.
`i~'`30 It is ~o be noted that although the intensity of
`~ the fluorescence emission of wavelength ~ 1~ derived
Y~ from the pH-insensitive fixst indicator, is itself
5~ independent of the pH of the medium, the fact that this
intensity is affected by energy absorbed by the second
~` 35 indicator, which is pH-sensitive, means that the ratio
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~ 32~772
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derived from the peak of the emission spectrum curve of
the first indicator and the isobestic point between the
two emission curves (as described in detail hereinafter
with reference to the drawings) also may be used to
give an accurate, stable determination of the pH of the
liquid medium.
The sensor system of the second embodiment of the
` invention preferably -ncludes an optical fiber having a
distal end and a proximal e~d, in which said combination
of fluorescent indicators, bicarbonate solution and
membrane is attached to said distal end and said
proximal end is adapted to receive excitation radiation
` from said source of excitation radiation.
As in the first embodiment, the preferrad first
^ 15 fluorescent indicator is 6,7-dimethoxycoumarin or a
pH-insensitive coumarin derivative, with 6,7-dimethoxy-
coumarin being particularly preferred.
Also the particularly preferred second fluorescent
indicator is HPTA.
`~` 20 In a particularly preferred form of the second
;;~ embodiment the 6,7-dimethoxycoumarin is directly bonded
~ to the distal end of an optical fiber and HPTA is
x suspanded in a gel of carboxymethyl cellulose
i~ contain~ng a bicarbonate solution, preferably aqueous
!,~ 25 sodium bicarbonate solution, which gel is bounded by a
~; silicone rubber membrane.
The method of the second embodiment is preferably
carrisd out by transmitting excitation radiation having
a wavelength ~ o, of 337 nm from a nitrogen gas laser
;~-
through the optical fiber from its proximal end to its
~,~ distal end where it excites the 6,7-dimethoxycoumarin
to emit fluorescent radiation having a wavelength ~ 1
of 435 nm. This fluorescence emission, in turn,
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excites the HPTA to emit fluorescent radiation at a
wavelength, ~ 2~ of 510 nm.
. When the sensor is immersed in a liquid medium
containing carbon dioxide, the latter permeates through
the silicone rubber membrane and reacts with the
bicarbonate solution thereby altering the pH of the
solution around the sensor. The intensity of the
fluorescence emission of wavelength ~ 2 (512 nm~ is
dependent upon the intensity of the excitation emission
of wavelength ^~ 1 and upon the pH of said surrounding
solution. Therefore, measurement of the ratio of the
~. .
intensities of the emitted radiation of wavelengths
2 provides an indication of the solution pH
within the membrane and thus a highly accurate, stable
determination of the concentration of carbon dioxide
~ (pCO2) in the liquid medium.
,; The accompanying drawings comprise graphs
illustrating excitation and emission spectra of the
,
indicators used in the sensors of the invention.
Figure 1 illustrates excitation spectra for HPTA
~;~ at varying pH levels.
Figures ~ illustrates pH-insensitive excitation
~`:!` and emission spectra of dimethoxycoumarin in a solution
~; of bicarbonate and ethylene glycol.
Figure 3 illustrates spectra for HPTA in ethylene
, .y
glycol at different pH levels.
Figure 4 illustrates spectra for HPTA in diferent
solvent mixtures.
Figure 5 is a graph showing HPTA fluorescence as a
~;~ 30 function of the water content of the system.
0 . . . .
~; Flgure 6 and Flgure 7 lllustrate spectra
indicating PCO2 by a sensor system according to the
invention.
Figure 8 illustrates spectra for varying carbon
dioxide concentrations using HPTA in ~ 50/50 ethylene
` glycol/water solution.
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Figure 9 is a graph showing the relationship
between the ratio of fluorescence intensity and carbon
dioxide concentration.
The excitation spectra for HPTA illustrated in
Figure 1 of the drawings taken over a wavelength range
of 300 to 485 nm show that the intensity of the
excitation radiation, which is a function of the area
; under the curve and is proportional to the height of the
,~ curve in each case, varies according to the pH of the
surrounding medium. In this case the pH was varied
from 6.66 to 8.132. Isobestic points were observed at
~, 337 nm and 415 nm. The peak wavelength of the emission
from HPTA subjected to the said excitation radiation
~ was 510 nm (not shown).
,i; 15 Figure 2 of the drawings illustrates excitations
and emission spectra for dimethoxy coumarin in a
solution of sodium bicarbonate and ethylene glyool.
The concentration of dimethoxy coumarin is about 10 2M.
The excitation spectrum exhibits a peak at a wavelength
of about 340 nm and the emission spectrum has a peak at
~ a wavelength of about 427 nm. The emission
;.~ fluorescence is pH insensitive. It will be noted that
~: the wavelength of the fluorescence emission for
dimethoxycoumarin overlaps the wavelength of the
excitation radiation for HPTA as illustrated in Figure
::~: 1.
Figure 3 illustrates spectra for HPTA in ethylene
glycol at pH 8.0 and pH 4.0, respectively. The HPTA is
dissolved in ethylene glycolr one drop of pH 8.0 buffer
is added and the solution is irradiated from a nitrogen
;~ laser with radiation of wavelength 337 nm. Two
fluorescent emissions at wavelengths 440 nm and 510 nm
~- are observed. One drop of p~ 4.0 buffer is then added
~i~ and the intensity of the spectra changes a~ illustrated
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in Figure 3. A peak at 510 nm appeared with the
addition of water to the system, regardless of the pH.
~ Figure 4 illustrates spectra of HPTA in different
,- mixtures of ethylene glycol and water. 10 M HPTA was
dissolved in solution mixtures comprising, respectively,
' 100% ethylene glycol, 80~ glycol/20~ water and 50%
~; glycol/50% water. Drops of each solution in turn were
put on the tips of optical fibers and the HPTA was
excited to fluoresce at a wavelength of 510 nm. The
results are shown graphically in Figure 4.
~` Additional results were obtained in a similar
~i manner for solutions comprising 20% glycol/80~ water
and 100% water. The results for all runs are given in
the following Table I.
,s .
. 15 Table I
Relative Relative
-~ Intensity Intensity
~I(Blue) ~(Green)
Solvent i!- 440 nm ~ = 510 nm Ratio _ l/Ratio
100 % ethylene 85.63 11.66 7.34 0.1362
` glycol
80/20 69.97 49.64 1.41 0.70g2
glycol/water
50/50 16.33 92.30 0.177 5.65
glycol/water
~ 20/80 5.33 83.30 0.064 15.63
'~ glycol/water
100% water5.33 111.96 0.04B 20.83.
The fluorescence of HPTA as a function of the water
content of the solvent system is illustrated
graphically in Figure 5. Using excitation radiation of
~ wavelength ~ = 337 nm the xatio of the fluorescence
; peaXs I(GREEN)/IIBLUE) at ~ ~ 510 nm and ~ _ 440 nm,
: respectively, was graphe~ for HPTA in ethylene
glycoltwater solutions of varying concentrations. T~e
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" -15- 1326772
resulting graph indicates that the ratio of intensities
increases substantially linearly as the water content
of the solution increases.
Figures 6 and 7 illustrate results obtained by
performing the invention as illustrated in the
following Examples.
Example_1
A mixture of 1:1 dimethoxycoumarin:HPTA both at a
concentration of 10 3M was dissolved in carboxymethyl-
~- 10 cellulose (CMC) and 5mM of sodium bicarbonate with the
addition of 0.25 ml ethylene glycol to dissolve the
dimethoxycoumarin.
A carbon dioxide sensor was formed by depositing
~`~ the resulting gel on the tip of an optical fiber formed
~ 15 by fused silica having a diameter of 400 m, and
;'. enveloping the gel in a carbon dioxide permeable
`~ silicone rubber membrane.
~ The sensor was irradiated with excitation radia-
. .
tion of wavelength 337 nm from a nitrogen laser firing
at 2 pulse/second. The fluorescence emission was
detected with a linear array photodiode and monitored
:~` with an oscillo~cope set to 0.1 volt/div. at 2 ms/div.
'~l A number of runs were conducted at varying carbon
dioxide concentrations and the results for 0~ CO2 and
100% CO2 are illustrated graphically in Figure 6 and
are set out numerically in the following Table II.
: Table II
~ % CO~ I (Blue~ I (Green) _ Ratio
:'t.,~' 0 5.~6 53.9~i 9.53
~ 30 100 30.32 20.99 0.69
~J:~
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Example 2
A gel containing mixture of 1:1 dimethoxy-
coumarin:HPTA at a concentration of 10 3 in CMC and 5mM
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~32~72
; of sodium bicarbonate was made up in a similar mannerto that described in Example 1 and this gel was used to
form a carbon dioxide sensor also as described in
Example 1.
A number of runs were conducted at varying carbon
dioxide concentrations and the results are illustrated
graphically in Figure 7 and set out numerically in the
' following Table III.
~` 10 ~ CO Baseline B lTnbe IM (COUM) IM (HPTA) Ratio
2 (COUM) (HPTA) (HPTA/COUM)
,.,
.~` 0% 9.5 10.5 20.99 150.93 12.22
y 7~ 9.5 10.5 40.65 97.29 2.79
~'100~ 8.5 10 78.~3 57.64 0.68
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lS The results given in the above Examples show the
accuracy with which quantitative results can be
~ obtained using the sensor system according to the
-'` invention.
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Example 3
This Example illustrates a carbon dioxide sensor
utilizing the relationship between the water content of
the system and the carbon dioxide concentration. HPTA
was dissolved in a 50/50 mixture of ethylene glycol and
water and the solutio embedded in a
carboxymethylcellulose gel. This gel was deposited on
the tip of an optical fiber and enveloped in a carobn
dioxide permeable silicone rubber membrane to form a
carbon dioxide sensor.
~' The s~nsor was irradiated with radiation of
~- 30 wavelength 337 nm from a nitrogen laser at varying
~ concentrations of carbon dioxide. The results are
`~ shown in Figure 8.
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~ It will be seen that the ratio of intensities of
- the fluorescence emissions at peak wavelengths of
460 nm and 510 nm is dependent upon the carbon dioxide
concentration. The spectra exhibit an isobestic point
at 485 nm.
The fluorescence ratio as a function of carbon
dioxide concentration is illustrated in Figure 9.
This Example illustrates the way in which a carbon
dioxide determination can be obtaiend as a function of
,~ 10 the water content of the sensor.
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