Note: Descriptions are shown in the official language in which they were submitted.
~116987
This invention relates to a process and an apparatu~ ~or
measuring the concentration o~ gaseous oxygen, carbon monoxide
and/or carbon dioxlde in gaseous or liquid samples, in par-
ticular in breath and blood samples.
In medical and scientific research as well as in other
flelds, for example in control measurements regularly carried
out to prevent environmental pollutlon or in rermentation
apparatus or in air, space or divlng technology, the problem
~requently arises o~ measuring small concentrations o~ oxygen,
carbon monoxide or carbon dioxide in small samples rapiclly and
su~ficlently accuratelyO
Numerous gas analytical processes based partly on I)hysical
and partly on chemical methods have long been known. P~lysical
determination Or oxygenJ carbon monoxide and carbon dio~;ide can
be carried out by mass spectrometry and gas chromatography.
Oxygen may in addition be determined by parama B etic ancl polaro-
graphic processes as well as by thermal conductivity measure-
ments and by means of electrol~tlc cellsO For carbon monoxide
and carbon dioxlde, additional processes ~ased on ln~ra-red ab-
sorption are availableO Conventional chemical processes lnclude
the manometric and volumetric methods Or oxygen and carbon
dioxide and volumetric methods o~ oxygen and carbon dloxide
determlnation (measurement o~ a change ln pressure or volume
after absorption of a gas ~raction) and the use of redox
systems for detecting the presence o~ oxygen or the recording
o~ colour changes which take place in haemoglobin when lt reaot
with oxygen or carbon monoxide.
Each one of these conventional methods has at least one
Or the followlng disadvantages: hl~h cost o~ apparatus, sus-
ceptibility Or the apparatus to break-down or malfunctioning,
Le A 17 ~79 - 1 -
~.
difficulty Or uslng the apparatus~ long time required ror
analysis, large sample volumes requlred, lack Or specificlty
for the gas under~investigation, measurement elther only in
the gaseous phase or only in the liquid phaseJ or the necessity
of using numerous and expenslve chemicals.
It is an ob~ect of the present lnvention to provide a
process which can be adapted to the indlvidual purpose ~d is
suitable for both gas and llquid samples and is employecl in
the same manner for three different gases and in whlch only
inexpenslve cheml¢als need to be replacedO At the same time,
lt is intended to shorten the time requlred for analysis and
to increase the sensitivity of the process.
According to the invention, there is provided a process
for the determlnation of the oxygen, oarbon monoxide and/or
carbon dioxlde gas contents of gaseous or liquld samples,
whereln the sample ls continuously supplied and mixed with a
reaction solution which is speci~lc ~or the gas to be analysed
and whlch undergoes a characteristic colour change in the
presence of thls gas, whlle maintaining a constant rate Or
supply and proportion Or sample to reactlon solutlon in the
mixture, and the concentratlon o~ the 2J C0 or C02 gas ls
determlned by measuring the degree Or absorptlon in a photo-
meterO
There is also provided a process for the determinatlon
Or the oxygen, ¢arbon monoxide and/or carbon dioxlde gas
content o~ gaseous or liquid samples, wherein the sample is
inJected from an lnJection syringe into an in~ection chamber
which is rilled with an inert liquid; and through whlch flows
a continuous stream Or a highly purified inert gas, the mixture
o~ the gas ~rom the gaseous or llquid sample and the inert gas
~ 2 - 2 -
5~7
is continuously supplied and mixed with a reaction solution
which is specific for the gas to be analysed and which under-
goes a characteristic colour change in the presence of this gas,
while maintaining a constant rate of supply and constant pro-
portion of sample to reaction solution in the mixture, and the
concentration of the 2' C0 or C02 gas is determined by measur-
ing the degree of absorption in a photometer.
There is further provided a process for the determination
of the oxygen, carbon monoxide and/or carbon dioxide gas con-
tents of gaseous or liquid samples, wherein the sample is in-
jeeted from a precision injection syringe into a transparent
test euvette which is closed on all sides and filled with a
reaction solution which is specific for the gas to be analysed
and which undergoes a characteristic colour change in the
presenee of the particular gas, and the concentration of the
2' C0 or C02 gas is determined by measurement of the degree
of absorption in the cuvette which is inserted in a photometer
after the expiry of a certain reaction time, the change in the
degree of absorption being determined by eomparison with a
standard euvette whi.eh is filled with the same quantity of
liquid but without the reagents used for the test.
In aceordanee with the present invention, the deter-
mination of oxygen consists of hydroxyl or amino derivatives of
aromatic eompounds or of derivatives of multinuelear aromatie
eompounds eontaining heteroatoms, the reaetion solution for the
determination of carbon monoxide comprises a solution of fuchsin
and hydrazine and the reaction solution for the determination
of carbon dioxide comprises a solution of fuchsin and hydrazine
hydrate.
The determination of oxygen is carried out by using a
'''`1''~ .
,~
lli6987
reaction mixture which yields an intensive colouration with
oxygen, preferably by using hydroxy- or amino derivatives of
aromatic compounds, e.g. benzene or naphthalene or derivatives
of polycondensed heteroaromatic systems~ Colour intensifying
substances, preferably metal salts, may be added, e.g. ferrous
ammonium sulphate (Mohr's salt). The effective component are
the Fe ions in this case.
For determining the oxygen concentration, it has been
- 3a -
D
round particularly suitable to use a reaction solutlon conD
taining rrom 5 to 100 mmol/litre Or pyrocatechol, rrom 1 to
20 mmol/litre Or ferrous ammonium sulphate (Mohr's salt) an
rrom 0002 to 5 N sodium hydroxide solution.
The photometric determinatlon o~ the reaction products is
carried out in the system pyrocatechol + Fe2+ at a wavel.ength
Or 490 nm, whereas in the case Or pyrogallol as reactant a
wavelength Or 520 nm is prererred~
The carbon monoxlde contents of blood can be easily
determined by using a reaction mixture, which is obtained by
diluting the blood with distilled water in proportions Or
between 1 : 200 to 1 : 2000. The photometer wavelength ror
the carbon monoxide determination is preferably 5~6 nm when
the blood concentration is near the upper limit (1 : 200) arLd
420 nm for a concentration near the lower llmlt (1 : 2000).
The reaction mixture used ror determining carbon dloxide
is preferably a solution o~ hydrazine hydrate with a concentrat-
ion o~ 1 to 10 mmol/litre and ~uchsin wlth a concentration o~
0.05 to 0.5 mmol/litreO The photometric wavelength is option-
ally at 545 nm in this case~
In the first mentioned prooess, there may be used a pump
Or known kind in which the sample Or gas or liquid together
with one or more suitable reaction solutions is ¢ontinuously
pumped to the place where it is to be mixed whlle the components
are maintained in a conætant proportion to each other, and the
mixture is tr~1sported to the photometer after the separation
o~ gas bubbles. When transporting a liquid sample in this way~
lt is advantageous to introduce bubbles Or an inert gas, in
particular highly puri~ied nitrogen, continuously into the
~0 stream Or reaction mixture. Furthermore, be~ore introducing
Le A 17 91~ _ 4 ~
~S~6~87`
a gas sample into this process, a zero ad~ustment should be
carried out in the photometer by passing an inert gas3 in
particular highly purified nitrogen, into the stream ~lowlng
through the dellvery channel and through the photometer.
Equalisation o~ pressure in the contalners ~rom whlch
the reaction solutions are delivered can be established by
washing out with a stream o~ an inert gas, in particular
nitrogen, which has previously been passed through an al~allne
pyrogallol solutionO
In the second modified process, an inJection chamber
may be used which is traversed by a stream of inert gas enter-
ing from the underside through a gas permeable and liquid
impermeable barrier, in particular a fritO This lnJection
chamber is advantageously connected to a liquid filled-
reservoir by means of a conne¢tlng pipe equipped with a
shutt-off valve.
The composition of the liquld depends on the kind of the
sample to be investigated and on the klnd of the gas. For
the ~inyes ~ ga~on of b~ ~d potasslum hexacyanorer ~ ~ (III)
is used for expelling thë oxygen, whereas carbondioxide is
expelled by dilluted acetic acld.
The quantitative proportlon o~ the gas to be investigated
is calculated by planimetric measurements o~ the surface
area under the graph in the curve tracer of the photometer
or by means of an electronic lntegratorO
In the third modified process, cuvettes which are
gas-tightly sealed with a lid made of a material which is
gas-tight but can be perforated are filled with the reaction
solution used in the particular test.
Oxygen concentrations are measured by means of a test
Le A 17 ~ 5 _
~169~7
cuvette sealed by a membrane which is impervious to oxygen
but can be perforated. This cuvette is partly filled with
inert gas and partly filled with a reagent which undergoes
a colour change in the presence of oxygen.
This cuvette is made of a gas impermeable material,
preferabl~ glass, and has a suitable form for photometric
measurements, e.g. it may be cylindrical (circular cuvette).
The following dimensions may be recommended for such a
cylindrical cuvette: Internal diameter lO mm (corresponding
to a layer thickness of about lO mm), external diameter
12 mm, capacity 3 to 5 ml.
The cuvette described above is preferably sealed by a. ~embrane
which is impervious to oxygen but can be perforated. The
membrane is composed of several layers of an elastic support
material such as rubber, synthetic high molecular weigh1;
polymers and gas-tight metal foils glued together, ior
~x~mple in the following arrangement: Synthetic high molecular
weight polymer - aluminium foil - rubber - aluminium foil -
synthetic high molecular weight polymer. The cuvette is
sealed by this membrane in a suitable manner, ior example
by glueing the membrane to it or by fixing it with a metal
ring.
The sample of which the oxygen content is to be measured
is injected from an injection syringe, especially a precision
syringe, into the cuvette by perforating the membrane.
After removal Df the syringe, the elastic foils o$ the
membrane ensurle that the membrane will remain impervious to
oxygen for several hours. This enables the measurement
(see below) to be carried out several hours after injection
of the sample.
Le A 17 979 - 6 -
~,
, . . .
ill6987
The oxygen concentration measurement described is
extremely simple to carry out. Since all that is required
is to inject a 10 to 100 pl sample from a precision syringe
into the cuvette and to read oif the absorption change in a
photometer, this test can be carried out by unskilled
operators Moreover, measurement oi the oxygen concentration
takes very little time, at the most 1 minute irom introducing
the blood sample to obtaining the result.
Due ~o the presence Or an alkaline medium in the
reaction solution mixture, any cells present in samples
of biological material (for example erythrocytes in full blood
samples for oxygen determination) undergo lysis and the
cell membranes dissolve. Other organic corpuscular
constituents~ for example those found in river water (algae
or the like) are also dissolved. If desired, this process
of solution may be facilitated by the addition of detergents,
e.g. 1 to 3% sodium dodecyl sulphate. Any interference
with the photometric measurement by corpuscular impurities
in the samples under investigation is thereby greatly
reduced or completely suppressed.
Due to the speed with which the measurements are
carried out and the fact that no biological reactions such
as respiration or the like can take place in the reaction
solution mixture used for the colour reaction, the measure-
ments oi oxygen concentration can also be carried out in
those biological systems in which the oxygen concentration
would be greatly altered by biological processes if measure-
ments took too long so that no significant results could be
obtained.
By diluting the sample (10 to 100 ~1) to the volu~e
Le ~ _ 7 _
;98~
of the reaction solution mixture, i.e. to about 2.5 ml, even
intensely coloured samples (e.g. blood) can be measured, since
as a result of the high dilution, the intrinsic extinction of
samples is relatively small compared with the extinction produced
by the presence of the oxygen.
This oxygen determination can be carried out both in
gases and in liquids. When measuring the oxygen content of
liquid samples, extraction or elution of the gas from the liquid
phase, which is normally necessary, can be dispensed with. The
equalisation of pressure which is necessary when injecting liquid
samples can be achieved by the presence of from 1 to 2 ml of
inert gas in the cuvel:te, corresponding to about 30 to 40% of the
total volume.
Measurement of the oxygen concentration can be carried
out in a mobile unit if a portable, battery operated photometer
is used for the absorption measurements. Diagnosis of the oxygen
concentration of human blood can therefore be carried out on the
spot, for example, in emergency and accident cases (medical
ambulances), in diving, air travel and space travel medicine
(submarines, aircraft) and in sports medicine and industrial
medicine.
In the accompanying drawings,
Figure 1 shows schematically an apparatus for carrying
out the first process,
Figure 2 shows an apparatus for carrying out the
modified second process,
Figure 3 shows a test cuvette for use in the third
process, and
Figure 3a shows the make-up of the membrane 34 of
Figure 3.
In the first process, represented schematically in
Fig. 1, a gas sample is delivered to a mixing spiral 12 by a
8 --
~1~6987
precision pump ll. The same pump also delivers reaction solution
I from vessel 13 and/or II from vessel 14 at a certain constant
ratio into the mixing spiral 12 where the colour change takes
place. After escape of the gas bubbles, the liquid sample is
passed through a photometer 15 where it is analysed in known
manner.
- 8a -
a~
1~'i6987
The zero value of the photometer may ~e determined by rirst
passing hlghly puri~ied nitrogen which has been aompletely ~reed
from oxygen in the wash bottle 16 containing an alkallne
pyrogallol solution through the apparatus for some time
in place of the gas sample before analysis is carried out.
The modified second process illustrated in Figure 2
is used mainly when only small quantities of sample are
available. The samples are taken up with a syringe 21 and
injected into a vertical injection chamber 22 ¢ontaining a
liquid. A stream of highly purified nitrogen entering
the lnjection chamber from below through a frit bottom plate
2~ flows through this liquid and agitates it so that
the gas to be measured in the gas sample or liquid sample
is carried upwards with the stream and delivered to the
precision pump 11.
This pump, in the same way as in the first process,
continuously delivers the reaction solutions in constant
proportions into the mixing spiral from which the coloured
solution is transferred to the photometer 15, The llquid
in the injection ohamber can be replaced by liquid from a
reservoir 26 by opening the tap 25 in!a conneotlng pipe 24
or it can be le!t o~ through a diæcharge pipe 27 by opening
the tap 28.
The third process merely require~ a precision
injection syringe, a test cuvette closed on all sides
and capable of being perforated on one side, which cuvette
contains one o~ the various reaction solutions, and a
photometer into which the test cuvette is irlserted a~ter
lnJsction of tlle sample æhaking and a short time o~ walting
(20 - ~0 sec), and in which the cuvette is inspected. The
Le A 1~ ~ 9 _ 9 _
~1~6~87
amount of change in the degree of absorption is obtained
by comparing the cuvette with a standard in the form o~ a
cuvette which is filled with the same quantity of liquid
but without the reagents used for the te~t. This proce~
thus does not operate continuously with the aid of a
precision pump but intermittently. An example of a test
cuvette ~or measuring oxygen concentrations is shown in
Figure 3. 3/
C This is a cylindrical glass cuvette-~, in which
the length of the light path is lO mm. The cuvette contains
2.5 ml of reaction liquid ~2;the remaining space in the
cuvette is filled with inert gas33; The cuvette 31 is
closed by a membrane~4 composed of several layers.
The membrane is pressed against the top surface of the
cuvette by a ~Icrew fitting 35. The sample is injected
through the membrane from an injection syringe. ~he
membrane (Fig. 3a) is composed o~ the following layers:
polyethylene ~6~aluminium ~7,Para rubber 38,aluminium 39,
polyethylene 310.
The advanl;ageSor the proCesses descrlbed above are
obvious. They enable the same apparatus to be used ~or
determining three dir~erent gases, simply by replacing a
~ew reagentsO The prooesses are so slmple that they can
be carried out by unskllled operators. The high sensitivitles
also play an important part. For the rirst des¢ribed process
the followlng maxlmum sensitivitles are obtained ln a stream
of gas sample at 2.5 ml per minute: Oxygen 0.002 vlume ~,
carbon monoxlde 00005 volume ~ and carbon dio~lde 0.01
volume %. A re!l~tlvely 8imple photometer may be uRed ~or
3o this process. In the second process~ the lower limlt Or
Le A 17 97~ - 10 -
1 116987
detection is Ool microlitre for oxygen, 005 mlcrolltre for
carbon monoxlde and 1 mlcrolitre ror carbon dioxide (under
standardphysical conditlons), using a relatively slmple
photometer.The third process is particularly suitable when
only small quantities of sample are availab~e and can be
used for gas or liquid samples measuring from about 1 to
100 microlitres~ Each sealed cuvette, for example, may be
filled with 2.5 millilitres of liquid and 1.5 millilitres
of inert gas. The length of the li~ht path in the cuvette
may be 10 mm so that the cuvette can be used ln any holder
of a conventional photometer. For medical diagnostic pur-
poses, thls means that venous punctures are not requlred
for obtainlng the blood sample (medical ancillarles can
take the necessary blood sample frOm the ear lobe), or
that oxygen ~termination can be carried out even when
only small quantities Or blood are availabe (determination
of the oxygen ¢oncentration in the blood taken from the
scalp in infants)0
In contrast to other processes, this process does not
measure the oxy~en partial pressure but the oxygen concen-
trationO This ls Or considerable dlagnostlc importance
for numerous cases, for example in poison cases in whioh
methaemoglobin is formed or in carbon monoxl~e pois~ning.
Another noteworthy feature of the process is the
simplicity of the apparatus requlred, which is re~lected
in low initial costs &s well as in low runn$ng costs since
only inexpensive chemicals are used. This simplicity in
the apparatus also makes lt very mobile so that the third
process, in particular, can also be used in mobile ~ield
units for environmental monitoring and control operations.
Le A 17 979
Other important ractors are the hlgh degree o~ variability
and adaptability to the given purpose resulting rrom the ~act
that three processes are available which oover all the possi-
bilities o~ gaseous or iiquid samples, large or small quantitles
of samples and three di~erent types o~ gasO
In all processes, moreover, the analysis tlme is reduced
to 2 minutes.
Le A 17 ~ 12 -
