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Description  |
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BACKGROUND OF THE INVENTION
This invention relates to a method for the fluorometric determination of
the concentrations of substances, such as gases or liquids, contained in a
sample, using fluorescent indicators whose fluorescence intensity is
reduced or quenched by the substances to be tested. The invention further
relates to an apparatus for implementing this method, the apparatus
including a sample chamber provided with fluorescent indicators in which
the sample containing the test substances is placed, the fluorescence
intensity of the fluorescent radiation which is excited in the above
indicators by a light source being diminishable or quenchable by the
substances to be tested.
An object of the present invention is to design a method and an apparatus
for the fluorometric determination of one or more substance concentrations
by means of fluorescent indicators which need no longer be specific to the
test substances, thus permitting different substances to be measured
simultaneously using fluorescent indicators that may be chosen
arbitrarily. A main object is the determination of concentration ratios or
other ratios of interest between two given concentrations.
DESCRIPTION OF THE PRIOR ART
It is known that the ability of certain dyes to fluoresce can be reduced by
the addition of certain substances, so-called quenchers. The relation
between the concentration of the so-called quencher and reduction in
fluorescence intensity, according to Stern and Volmer, is as follows:
##EQU1##
In this context, F.degree. denotes the fluorescence intensity of a
fluorescent indicator in the absence of a quencher,
F the fluorescence intensity of a fluorescent indicator in the presence of
a quencher,
k the so-called quenching constant specific to each pair of
quencher/fluorescence indicator,
[Q] the concentration of the quenching substance or quencher.
Since F.degree. and k are constants for the respective fluorescent
indicators or indicator substances used, the quencher concentration may be
inferred from the measurement of F. If F.degree. and k are unknown, they
are eliminated by performing measurements and by preparing calibration
lines or curves, F being plotted against [Q], and F.degree. and k being
determined graphically, for instance.
This analytical method has found various applications. In U.S. Pat. No.
3,612,866, for instance, there is shown a method for measuring oxygen
concentrations in various samples, e.g. blood. The method is based on the
ability of oxygen as a quencher to reduce the fluorescence intensity of
certain aromatic hydrocarbons, e.g. pyrene, in accordance with equation
(1). In the journal "Biochemistry" (vol. 9, p.464, 1970) a method is
described for tracing the local oxygen concentration by observing the
quenching of fluorescence by means of pyrene butyric acid. In "Zeitschrift
fur Analytische Chemie" (vol. 314, p. 577, 1983) a method is described for
determining with great accuracy the concentration of halide ions by using
certain heterocyclic fluorescent indicators and observing the ensuing
quenching of fluorescence. German Laid Open print No. 25 08 637 shows a
method for the optical measurement of blood gases which is also based on
the principle of fluorescence quenching. Other analytical methods for
determining the concentrations of certain substances by means of
fluorescence quenching are described in the journal "Analyst" (vol. 107,
p. 465, 1982).
All of the above methods are characterized by a common disadvantage: The
fluorescent indicators or indicator substances must be specific to the
substance to be measured, i.e., they must only respond to this particular
sample substance. In practice, most fluorescent indicators will respond to
more than one substance, however, or rather, they will be quenched by a
number of substances.
SUMMARY OF THE INVENTION
The method described by the present invention enables for the first time
simultaneous fluorometric determination of several analytical quantities
or substance concentrations, even if the indicator is no longer a specific
one--, or determination of one single concentration despite the presence
of interfering or quenching substances. According to the present invention
this is achieved--above all, if the concentrations of two or more
substances are to be determined simultaneously--by performing several
measurements of fluorescence intensity on the sample containing these
substances, the number of measurements corresponding to the number of
substance concentrations to be determined, using at least one fluorescent
indicator which is non-specific relative to at least one of the substances
to be measured or which may be quenched by more than one of the
substances, and which has different quenching constants with regard to the
individual substances ("quenchers") quenching its intensities, and by
obtaining the concentrations of the individual substances and/or the
ratios of substance concentrations from the known, unquenched fluorescence
intensities of the fluorescent indicators employed, and from the quenched
or reduced fluorescence intensities obtained by measuring, and the various
quenching constants which are known or have been obtained beforehand by
graphical or computational methods.
The invention is based on the finding that equation (1) must be extended in
order to reflect mathematically the influence of several quenchers, by
adding more terms representing the influence of additional quenchers,
i.e., additional substances to be measured. There results an equation for
the case of two quenchers (2), and a general equation (3) for the instance
of several quenchers or test substances quenching non-specific
fluorescence indicators
##EQU2##
As equation (2) has two unknowns and equation (3) several unknowns, i.e.,
the various substance or quencher concentrations [Q], two or more
independent measured values are required for solution; in the general case
n measured values. They are obtained by measuring the fluorescence
intensity F of two different fluorescent indicators quenched by the
substances to be measured.
If an indicator A is used, a measured signal
##EQU3##
will be obtained.
In equation (4),
F.sub.A .degree. is the fluorescence intensity of indicator A in the
absence of quenchers or the substances to be measured;
F.sub.A is the fluorescence intensity in the presence of quenchers or the
substances to be measured;
.sup.1 k.sub.A is the quenching constant of indicator A given quencher
Q.sub.1 ;
.sup.2 k.sub.A is the quenching constant of indicator A given quencher
Q.sub.2 ;
[Q.sub.1 ] and [Q.sub.2 ] are the concentrations of the two quenchers.
If indicator B is used, the equation reads:
##EQU4##
F.sub.B .degree. is the fluorescence intensity of indicator B in the
absence of quenchers;
F.sub.B is the fluorescence intensity in the presence of quenchers;
.sup.1 k.sub.B is the quenching constant of indicator B given quencher
Q.sub.1 ;
.sup.2 k.sub.B is the quenching constant of indicator B given quencher
Q.sub.2 ;
[Q.sub.1 ] and [Q.sub.2 ] are the concentrations of the two quenchers.
If, for the sake of simplicity, .alpha. is substituted for
##EQU5##
and .beta. is substituted for
##EQU6##
and if [Q.sub.2 ] calculated from equation (4) is put into equation (5),
the following expression will result:
##EQU7##
while for [Q.sub.2 ] one obtains:
##EQU8##
.sup.1 k.sub.A, .sup.2 k.sub.A, .sup.1 k.sub.B and .sup.2 k.sub.B are
constant quantities that are known or have already been obtained. .alpha.
and .beta. are variable quantities resulting from the values measured for
the quenched intensities F.sub.A or F.sub.B. From equations (6) and (7)
the concentrations of the test substances or quenchers Q.sub.1, Q.sub.2
may now be obtained.
If three substances or quenchers are to be determined, three fluorescent
indicators are necessary, and three quantities .alpha., .beta. and .gamma.
must be measured, which are defined as follows:
##EQU9##
Using equation (3) this will result in three equations with nine quenching
constants:
.alpha.=.sup.1 k.sub.A .multidot.[Q.sub.1 ]+.sup.2 k.sub.A
.multidot.[Q.sub.2 ]+.sup.3 k.sub.A .multidot.[Q.sub.3 ]
.beta.=.sup.1 k.sub.B .multidot.[Q.sub.1 ]+.sup.2 k.sub.B
.multidot.[Q.sub.2 ]+.sup.3 k.sub.B .multidot.[Q.sub.3 ] (9)
.gamma.=.sup.1 k.sub.C .multidot.[Q.sub.1 ]+.sup.2 k.sub.C
.multidot.[Q.sub.2 ]+.sup.3 k.sub.C .multidot.[Q.sub.3 ]
In matrix notation, this system of equations may be written as
##EQU10##
and may be solved with the use of a suitable pocket calculator.
For determination of n unknown concentrations, n independent equations are
set up in the same manner, and n values have to be measured for the
quenched fluorescence intensities F.sub.n.
The advantage of this method is that it will make it possible for the first
time to determine fluorometrically, for instance by means of two
non-specific fluorescent indicators, two unknown substance concentrations,
provided that each of the two substances will quench both fluorescent
indicators.
It will also be possible to determine only the concentration of one
substance in the presence of an irrelevant or an interfering substance
acting as a quencher. In this instance again two measured values will be
required, i.e., the quenched fluorescence intensities of the two
fluorescent indicators, the values for the substance concentrations being
calculated as described above, except for the fact that the quenched
intensity of the interfering substance is needed as an auxiliary variable
and that the actual concentration of the interfering substance need not
necessarily be calculated. In this case the method described by the
present invention will be used for discriminating the desired substance
concentration from that of an interfering substance, and will be
characterized in that, for determining the concentration of at least one
substance in the presence of at least one other, irrelevant or
interfering, substance quenching the fluorescence intensity (-ies) of the
fluorescent indicator(s) responding to the substance(s) to be measured,
the concentration(s) of the other, interfering substance(s) is (are)
determined just as the concentration(s) of the substance(s) to be
measured, and is (are) used for determining the concentration of the
substance to be measured.
Similarly, any effects of n interfering or irrelevant substances may be
eliminated by n measured values for quenched fluorescence intensities of
these unwanted substances, which, together with the quenched fluorescence
intensity of the substance to be measured, are put into n+1 equations. The
equations can only be solved if the quenching constants k are different
for all indicator/quencher combinations. The number of concentrations to
be determined thus is identical with the nunber of quenched intensities of
relevant and interfering substances necessary for solving the equations.
Equations (6) and (7) are greatly simplified by using at least one
indicator specific to a substance. In this case one of the quenching
constants will equal zero, and the fraction will consist of three terms
only.
If only the ratio of two quencher concentrations is to be determined,
equation (11) will apply which results from combining equations (6) and
(7):
##EQU11##
It may be possible that fluorescent indicators are used which are made
from the same fluorescent indicator substance, but that this substance is
maintained in different indicator environments, for instance in different
solutions or bonds and that these fluorescent indicators have different
quenching constants in the different indicator environments for the
substance or quencher to be measured. Fluorescent indicators or indicator
substances which are embedded in different environments and therefore have
different quenching constants varying with the respective substances used
should be regarded as different fluorescent indicators which are to be
used independently of each other for the method under discussion.
Depending on the individual application, fluorescent indicators may be used
which are in the same phase or contained in the same sample substance as
the substances to be tested. it may be of advantage if the substances to
be tested are allowed to diffuse into the indicator phase in case the
fluorescent indicators are in a different phase or are separated form the
test substances and/or if the fluorescent indicators are added
simultaneously to the test substances, or rather to the sample containing
these substances, or are distributed over several substance solutions or
indicator membranes. It will also be possible to have fluorescent
indicators which are in the same phase or same sample material as the
substances to be tested.
The choice of phase, i.e., whether the fluorescent indicator is to be in
the same phase as the test substance or in a different one, will depend on
the particular requirements. If the indicator is in the same phase, a high
measurement sensitivity may be achieved as the quenchers will be able to
reach the indicator practically unimpeded. On the other hand, quenching
specificity will be low since virtually all quenchers present in the
solution will become active. If the indicator is in a different phase this
will usually reduce measurement sensitivity as the quencher will first
have to diffuse into the other phase where its quenching efficiency will
be lower, at least in most practical cases (e.g. in a polymer), which will
result in smaller signal changes and thus in lower sensitivity and
accuracy. On the other hand, quenching specificity will improve markedly
since only substances able to diffuse into the second phase may act as
quenchers, i.e., mostly gases or small molecules only.
According to the invention, an apparatus as mentioned in the beginning for
implementing the method described is characterized in that the sample
chamber is provided with a number of fluorescent indicators corresponding
to the number of substance concentrations to be determined simultaneously,
and that at least one of these fluorescent indicators will respond
non-specifically to at least one of the substances to be measured, or will
be quenchable by more than one substance, and that the quenching constants
of this concentration indicator will vary for each of its quenchers, and
that the signals of the individual fluorescent indicators corresponding to
the reduced or quenched fluorescence intensities will be passed to an
evaluation unit, possibly via amplifiers, for determining the
concentrations and/or the concentration ratio of the indivdual substances
to be measured. This arrangement will permit the concentrations or
concentration ratios of substances to be determined with the use of
non-specific fluorescent indicators, even if these substances are mixed
with irrelevant interfering substances.
In a preferred form of the invention the evaluation unit is provided with a
divider unit into which are fed the values measured for the unquenched
fluorescence intensity of the individual fluorescent indicators as well as
the values for the indivdual fluorescence intensities obtained from the
fluorescent indicators, possibly after subtraction of a constant value
corresponding to a straylight component, and the resulting quotient is fed
into a subtraction unit contained in the evaluation unit where the value
of 1 may be subtracted from this quotient, and the above evaluation unit
is provided with a calculator unit for calculating the concentrations
and/or concentration ratios of the individual substances.
It will be convenient if at least some of the fluorescent indicators
contain an indicator substance which is maintained in a different
environment, solution or bond, for each individual indicator, and has
different quenching constants relative to its quenchers.
Depending on the respective requirements, it may be of advantage if the
fluorescent indicators or substances are distributed over several
substance solutions or indicator membranes, and/or if reaction spaces are
provided between the sample chamber and the fluorescent indicators, in
which quenchable substances can be transformed into nonquenchable
substances, or vice versa, by chemical reaction, and/or if the indicator
elements or fluorescent indicators are placed in the tip of a test probe
that may be dipped into the sample containing the substances. Apart from
the possibility of using the sample chamber as a reaction space at the
same time, a separate reaction space may be provided behind the sample
chamber.
In order to eliminate intensity variations of the light source it will be
convenient for evaluation purposes to provide the fluorescent indicators
with a reference light measuring cell or a reference indicator which may
be connected to the calculator unit via an amplifier.
DESCRIPTION OF THE DRAWINGS
The following is a more detailed description of the invention, as
illustrated by the enclosed drawings, in which
FIGS. 1-3 show various measuring arrangements, and
FIG. 4 presents a diagram for determining the quenching constants.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
FIGS. 1 and 2 show simple arrangements for implementing the method
described by the present invention for the common case of two diffusible
substances in liquid or gaseous samples. On the outside of a sample
chamber 20 fluorescent indicators or sensors 21A and 21B are provided. In
the fluorescent indicators 21A and 21B light sources 22A and 22B will
generate fluorescences whose intensities are measured by photodetectors
23A, 23B. In the absence of quenchers or the substances to be tested the
fluorescence intensity F.degree. will be particularly high; the measured
values will correspond to the quantitites F.sub.A .degree., F.sub.B 20 in
equation (2). For measurement purposes the sample containing the test
substances is introduced into, or passed through, the sample chamber 20.
From sample chamber 20 quencher substances may diffuse into the
fluorescent indicators 21A, 21B (e.g., oxygen, sulphur dioxide, halothane,
etc.). In the above indicators these quenchers will cause a reversible
reduction of the fluorescence intensity to the value F (equations (2), or
(4) and (5) ). If the fluorescence intensity F.sub.A, F.sub.B of the
indicators 21A, 21B is measured, the concentrations of the quenchers may
be calculated from the known values of F.sub.A .degree. and F.sub.B
.degree. and of the quenching constants k. For better discrimination of
the excitation light 24 and the fluorescent light 25, optical filters or
interference filters 26, 27 may be introduced into the radiation path.
The sample chambers 20 may be designed as single sample measurement
chambers or as flow measurement cells. The method and arrangement
described by the present invention may be used for measuring both single
samples and continous sample streams.
Light sources may include thermoelectric, electronic, laser- or LED-type
devices operating either in a continous or in a pulsed mode.
The fluorescent indicators used in this context usually consist of
solutions of quencher-sensitive materials or of indicator substances in
thin membranes made of polymer materials, containing plasticizers, if
required. As an alternative, fluorescent indicator materials or substances
may be covalently bonded to a carrier material.
Suitable indicators are fluorescent indicators or indicator substances,
possibly in differing environments, whose fluorescence is quenched by
extraneous substances. These indicators should be highly fluorescent and
stable. The following indicator/quencher combinations are given as
examples:
pyrenbutyric acid --oxygen
chlorophyll --sulphur dioxide
quinine sulphate --chloride ion
acridine sulphate --bromide ion
indole --H.sub.2 O.sub.2
benzo(ghi)perylene --haloethane
The photodetectors 23A, 23B are used for measuring the intensity of the
fluorescent light which will depend on the quencher concentration. For
light measurement or detection photoelectric cells, photoamplifiers or
photodiodes may be used. The measurement signal obtained usually is
amplified in an amplifier unit 28 before it is fed into an analog or
digital calculator or evaluation unit 29 and a display unit 30. In the
calculator unit 29, which can include a divider unit 29a, a subtraction
unit 29b and a calculator unit 29c, the concentrations of the substances
to be investigated are determined by means of the above equations.
FIG. 3 presents a schematical view showing the continuous measurement of
three substances in optically transparent material streams, e.g., of gases
or liquids. The test substances will flow in the direction of arrows 34
through a sample chamber 20 formed by an U-shaped pipe, passing three
fluorescent indicators 21A, 21B, 21C, which are excited to fluorescence by
the light 31 of a light source 22 that may have passed a filter 26. The
fluorescent light 32 emitted by the indicators 21A, 21B, 21C is measured
by detectors 23A, 23B, 23C, possibly after passage of light filtering
devices 27. The signals received are amplified in amplifiers 28 and are
evaluated by means of an evaluation unit 29 according to equations (8),
(9) and (10). They may then be displayed in the display unit 30 in an
analog or digital manner, or they may be printed out. The ratio of
quencher concentrations is determined with the use of equation (10).
It should be noted that it may often be practical to subtract a constant
quantity F.sub.s from the signal measured or from the measured
fluorescence intensity F in order to arrive at the measurement signal
proper. The quantity F.sub.s will help to take into account interferences
from the fluorescent light or stray light entering the fluorescence
detection system on account of light scattering. The stray light component
F.sub.s is determined, firstly, by modifying the Stern-Volmer-equation
such that the straylight component F.sub.s is taken into account:
Stern-Volmer:
##EQU12##
I.degree., I : detected light intensities (fluorescent light + stray light)
The result is:
##EQU13##
A three-point calibration will result in three calibration values:
##EQU14##
leaving two equations with two unknowns (k, F.sub.s):
##EQU15##
In this manner straylight components F.sub.s and quenching constants for
any sensor (any quencher) may be determined by calibration.
The description of these measuring arrangements should help to explain the
principle of measurement. In accordance with the special requirements of
any particular application the apparatus may vary and other components may
be used. It may become necessary, for example, to use optical fibres for
the transport of light from the light source to the fluorescent
indicators, and from the indicators to the photodetectors. Besides, it is
possible to add so-called ion carriers to the indicator membranes in order
to facilitate diffusion of the quenchers through the membranes and into
the indicators, or to enhance selectivity.
There are several ways of obtaining the desired measurement values or
concentrations. If two values are required, which is frequently the case,
(a) the fluorescence intensities F.sub.A and F.sub.B of two fluorescent
indicators containing different indicator substances may be measured in
the presence of the quenchers or the substances to be determined, upon
which the concentrations of the quenchers Q.sub.1 and Q.sub.2 can be
obtained by using equations (6) and (7), or by graphical methods;
(b) the fluorescence intensities F.sub.A and F.sub.A ' of two fluorescent
indicators may be measured, containing the same indicator substance, but
each in a different indicator environment, e.g., in different solvents or
different polymer membranes. As the quenching constants k of an indicator
substance will vary with the type of environment or solvent, the
concentrations of two quenchers again may be obtained by means of
equations (6) and (7), or by graphical methods. F.sub.B ' should be
substituted by F.sub.A '.
The same will apply if more than two materials or indicator substances in
more than two different environments or solutions are to be investigated.
The methods of measurement covered by the invention may vary in type,
setup and geometrical arrangement.
Interaction between fluorescent indicator or indicator substance and
quencher may take place in a homogeneous solution, by combining solutions
of the substance to be tested and the indicator (e.g., measurements in
cuvettes, as discussed in the subsequent examples 1 and 2).
Interaction between fluorescent indicator and the substance to be tested
may also take place by using indicators whose phase differs from that of
the sample substance, and by letting the quencher substances diffuse from
the sample containing the substances into the indicator or indicator
substance (e.g., when determining gas concentrations by means of
membrane-type sensors, as shown in FIGS. 1-3).
Fluorescent indicators which emit radiation permitting spectral
discrimination may be embedded in one single solution or membrane, or they
may be distributed over a number of solutions or membranes.
It is possible to place several membranes either one above the other or
side by side. In order to improve selectivity the indicators or indicator
elements may be covered by additional, permeation-selective membranes.
In another variant of the invention the indicator elements may be provided
with reaction spaces in which quenching substances are transformed into
nonquenching ones by means of chemical reactions, or, vice versa,
non-quenching substances are made quenching, thus permitting an indirect
measurement of non-quenching substances as well.
Fluorescent indicators in this context denote either indicator substances
as such or indicator substances applied to carrier media.
Intensity fluctuations of the light source 22 may be eliminated by
incorporating a reference light measuring cell 33 (FIG. 3) which will
measure the intensity of the light source 22 and is connected to the
amplifiers 28 in order to compensate fluctuations of the light source 22,
or rather, to avoid their being interpreted as changes in fluorescence
intensity. The present invention has a wide variety of applications, some
of which are mentioned below. The examples given should not be taken to
preclude other possible uses.
(a) Simultaneous measurement of oxygen and sulphur dioxide in industrial
waste gases, or in measuring facilities for environmental pollution.
(b) Measurement of oxygen concentrations, e.g. in road tunnels, in the
presence of interfering automobile exhaust gases (SO.sub.2, NO.sub.x).
(c) Simultaneous measurement of the quenchers oxygen and haloethane in
anaesthetic gas during anaesthesia.
(d) Continuous measurement of chloride and sulphite in effluents.
Following is detailed explanation of the invention by means of examples
demonstrating the accuracy of the method of measurement described by the
invention.
EXAMPLE 1
Quantitative simultaneous fluorometric measurement of two quenchers
(chloride and bromide) in homogeneous solution
Underlying principle: The fluorescence intensity of the indicators quinine
and acridine is quenched differently by bromide and chloride.
5.123 mg 1-chloro-2,4-dinitrobenzene (MW 202.56) and 4.602 mg
benzalacetophenone dibromide (MW 368.08) were weighed together and burnt
in an oxygen atmosphere. The combustion gases were absorbed in 5 ml of a
1-percent solution of hydrazine sulphate.
After absorption of the reaction gases the content of the flask was
transferred to a 20-ml-graduated flask which was filled up without the
addition of an indicator. 8 ml of this stock solution each were then
pipetted into two 10-ml-graduated flasks. After the addition of 1 ml of a
2.0 10.sup.-5 molar solution of quinine sulphate in 1 N sulphuric acid
(indicator 1) to one of these flasks, and of 1 ml of a 1.0 10.sup.-5 molar
solution of acridine in 1 N sulphuric acid (indicator 2) to the other
flask, both flasks were filled up with water to the 10-ml-mark. After
preparation of two halide-free standard solutions their standard
fluorescence intensities (F.sub.A .degree. and F.sub.B .degree.) were set
to 100. The relative F.sub.A value of the sample (indicator: quinine
sulphate) was 76.1, and the F.sub.B value of the sample (indicator:
acridine) also happened to be 76.1.
The quenching constants k necessary for evaluation of the test results had
been determined beforehand and are listed in Table 1.
TABLE 1
______________________________________
Quenching constants k for the indicators
quinine sulphate and acridine (as used in the
present example) in sulphuric acid.
Indicator Cl.sup.- Br.sup.-
______________________________________
Quinine sulphate in
.sup.1 k.sub.A =
.sup.1 k.sub.B =
0.1 N H.sub.2 SO.sub.4 (A)
133.0 177.5
Acridine in .sup.2 k.sub.A =
.sup.2 k.sub.B =
0.1 N H.sub.2 SO.sub.4 (B)
9.5 304.2
______________________________________
Results: The following test results were obtained:
F.sub.A .degree.=F.sub.B .degree.=100
F.sub.A =F.sub.B =76.1
.alpha.=(F.sub.A .degree./F.sub.A)-1=0.31506
.beta.=(F.sub.B .degree./F.sub.B)-1=0.31506
If the above quantities and the Stern-Volmer-constants k are entered into
equations (6) and (7), the molar concentrations of the chloride and
bromide quenchers are:
Cl.sup.- =1.0296 mM=0.9126 mg/25 ml total sample volume
Br.sup.- =1.0036 mM=2.0048 mg/25 ml total sample volume
The halide content in mg is obtained by multiplying the molar quencher
concentrations of the samples with the molecular weight and the total
sample volume in ml, and by dividing them by 1000.
If the measured value in mg/total sample volume is divided by the input in
mg, and the result is multiplied by 100, the relative content of halides
as a percentage will be obtained. For the present example this yields,
with a total input of 9.9725 mg, for chloride 9.38%, versus a calculated
9.22%, and for bromide 20.61%, versus a calculated 20.55%.
EXAMPLE 2
Quantitative simultaneous fluorometric measurement of three quenchers
(chloride, bromide, iodide) in homogeneous solution
Underlying principle: The fluorescence of the indicators quinine, acridine
and harman is quenched diffrently by chloride, bromide and iodide.
The simultaneous fluorometric measurement of three quenchers was tested by
means of three potassium halide solutions. First of all, the quenching
constants were determined and their additive combination into one overall
quenching constant was checked. The results of this measurement are
presented in Table 2.
TABLE 2
______________________________________
Ion specific quenching constants and overall
quenching constants for halides which are
simultaneously present in the solution.
Indicator: quinine sulphate in 0.1 N H.sub.2 SO.sub.4.
Overall halide concentration
mM halide k.sup.a
______________________________________
1 KCl 133
1 KBr 178
1 KJ 243
2 KCl + KBr 310
2 KCl + KJ 377
2 KBr + KJ 421
3 KCl + KBr + KJ
557
______________________________________
.sup.a The overall quenching constants refer to identical concentrations
of the quenchers present.
As is shown in Table 2, the overall quenching constants are additive
combinations from the individually determined ion-specific constants. This
proves that the contributions of the individual ions or substances to the
quenching effect are independent of the presence of other quenchers.
For simultaneous measurement of the three halides, 1 ml each of a 1.00
10.sup.-2 molar solution of KCl, KBr od KJ was pipetted into each of three
10-ml-graduated flasks. From the indicators quinine sulphate, acridine and
harman, solutions in 1 N sulphuric acid were prepared, and 1 ml of the
first of the three indicator solutions was added to the first flask, 1 ml
of the second solution was added to the second flask, and 1 ml of the
third solution was added to the third flask. After the graduated flasks
had been filled up, the fluorescence intensity F of the samples relative
to the fluorescence intensity F.degree. of the respective standard
solutions (1 ml indicator solution/10 ml water) was measured. Their
standard fluorescence intensity F.degree. was set to 100 and checked after
each measurement. The quenching constants found for the individual
indicators are presented in Table 3.
TABLE 3
______________________________________
Quenching constants for the simultaneous
measurement of chloride, bromide and iodide in
homogeneous aqueous solution.
Indicator Cl.sup.- Br.sup.- J.sup.-
______________________________________
Quinine sulphate in 0.1
133 (.sup.1 k.sub.A)
178 (.sup.2 k.sub.A)
243 (.sup.3 k.sub.A)
N H.sub.2 SO.sub.4 (A)
Acridine in 0.1 N H.sub.2 SO.sub.4
9.5 (.sup.1 k.sub.B)
304.1 (.sup.2 k.sub.B)
396.7 (.sup.3 k.sub.B)
(B)
Harman in 0.1 N H.sub.2 SO.sub.4
0.2 (.sup.1 k.sub.C)
9.4 (.sup.2 k.sub.C)
212.4 (.sup.3 k.sub.C)
(C)
______________________________________
The three values measured for the relative fluorescence intensities
F.sub.A, F.sub.B, F.sub.C were:
F.sub.A =64.3, F.sub.B =58.4, F.sub.C =81.8
F.sub.A .degree.=F.sub.B .degree.=F.sub.C .degree.=100 standard
fluorescence intensities
.alpha.=(F.sub.A .degree./F.sub.A)-1=0.55521
.beta.=(F.sub.B .degree./F.sub.B)-1=0.71233
.gamma.=(F.sub.C .degree./F.sub.C)-1=0.22249
Calculation according to equations (8) to (10) yields:
System determinant=7759322.2
Determinant or co-determinant 1=7762.839
Determinant or co-determinant 2=7786.451
Determinant or co-determinant 3=7776.161
For calculation of the i-th unknown the i-th column of the system
determinant was replaced by the left-hand side of equation (10). This
yields the following concentrations of the halide solutions:
[Cl.sup.- ]=1.000 mM
[Br.sup.- ]=1.003 mM
[J.sup.- ]=1.002 mM
The concentrations determined in this manner correspond closely to the
input values (1.000 mM).
EXAMPLE 3
Simultaneous continuous measurement of oxygen and halothane in an
anaesthetic gas
Underlying principle: The fluorescence of certain aromatic hydrocarbons is
quenched by oxygen and halothane (an anaesthetic gas with the symbol
2-bromine-2-chlorine-1,1,1-trifluoroethane).
In a measuring arrangement as presented schematically and in a simplified
form in FIG. 2, the quenching of fluorescence by means of a gas containing
20% oxygen and 4.4% halothane, was measured. The fluorescent indicators
21A and 21B contained solutions of decacyclene or benzo(ghi)perylene or
heterocyclic compounds in cross-liked silicones or polyvinylchloride with
a high plasticizer content. In preliminary tests--as shown in FIG. 4--the
quenching constants .sup.1 k.sub.A (quenching of fluorescent indicator 21A
by the first quencher O.sub.2), .sup.2 k.sub.A (quenching of indicator 21B
by halothane) .sup.1 k.sub.B (quenching of indicator 21B by O.sub.2 and
.sup.2 k.sub.B (quenching of indicator 21B by halothane) were determined
by experiment, using equation (1). In FIG. 4, the O.sub.2 /halothane
concentrations [Q.sub.1 ][Q.sub.2 ] are indicated along the abscissa axis,
and the quotient of standard and relative fluorescence intensity minus 1
is plotted along the ordinate axis. The gradients of the plotted lines
correspond to the quenching constants of indicator 1 for O.sub.2 or
halothane.
The quenching constants of indicators 1 and 2 are listed in Table 4.
TABLE 4
______________________________________
Quenching constants of fluorescent indicators
containing aromatic hydrocarbons, using oxygen
and halothane as quenchers.
______________________________________
Sensor 1 .sup.1 k.sub.A = 0.202
.sup.2 k.sub.A = 0.811
Sensor 2 .sup.1 k.sub.B = 0.771
.sup.2 k.sub.B = 0.643
______________________________________
The following values were obtained:
F.sub.A .degree./F.sub.A =1.073; therefore .alpha.=0.073;
F.sub.B .degree./F.sub.B =1.180; therefore .beta.=0.180.
If .alpha., .beta. and the quenching constants of Table 4 are inserted in
equations (6) and (7), the values obtained for the concentrations of
O.sub.2 [Q.sub.1 ] and halothane [Q.sub.2 ] will be 0.1999 and 0.043,
respectively, corresponding to 19.99 and 4.3 percent, respectively. This
is in excellent accordance with the actual values of the oxygen and
halothane contents.
* * * * *
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