GB1582376A

GB1582376A - Method for measuring the concentration of gaseous oxygen or carbon dioxide in gaseous or liquid samples in particular in breath and blood samples

Info

Publication number: GB1582376A
Application number: GB15011/77A
Authority: GB
Original assignee: Compur Electronic GmbH
Current assignee: Compur Electronic GmbH
Priority date: 1976-04-17
Filing date: 1977-04-12
Publication date: 1981-01-07
Also published as: SE8304989D0; JPS52154693A; SE456371B; SE441218B; FR2348490B1; SE8304989L; FR2348490A1; SE7704295L; CH632844A5; BR7702432A; JPS6136184B2

Abstract

To measure the proportions of gases, a reagent solution is used which is specific for the gas whose proportion is to be measured and which discolours in a characteristic manner if the gas in question is present. The sample and the respective reagent solution are fed in and mixed together while maintaining a constant ratio of the amounts fed in or the mixing ratio of the sample and the reagent solution. The concentration of the substance producing the colour (O2, CO or CO2) is determined by measuring the absorbance in a photometer. Such a method makes it possible to carry out the measurement for three different gases in the same way and requires inexpensive chemicals only to be replaced in the process.

Description

(54) METHOD FOR MEASURING THE CONCENTRATION OF GASEOUS OXYGEN OR CARBON DIOXIDE IN GASEOUS OR LIQUID SAMPLES, IN PARTICULAR IN BREATH AND BLOOD SAMPLES (71) We, COMPUR-ELECTRONIC GMBH, a body corporate organised under the Laws of Germany, of D 8000 Munchen 70, Germany, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to a method for measuring the concentration of gaseous oxygen or carbon dioxide in gaseous or liquid samples, in particular in breath and blood samples.

In medical and scientific research, as well as in other fields, for example in control measurements regularly carried out to prevent environmental pollution or in fermentation apparatus or in air, space or diving technology, the problem frequently arises of measuring small concentrations of oxygen or carbon dioxide in small samples rapidly and sufficiently accurately.

Numerous gas analytical methods based partly on physical and partly on chemical methods have long been known. Physical determination of oxygen or carbon dioxide may be carried out by mass spectrometry and gas chromatography. Oxygen may in addition be determined by paramagnetic and polarographic methods, as well as by thermal conductivity measurements and by means of eletrolytic cells. For carbon dioxide, additional methods based on infrared absorption are available. Conventional chemical methods include the manometric and volumetric methods of oxygen and carbon dioxide and volumetric methods of oxygen and carbon dioxide determination (measurement of a change in pressure or volume after absorption of a gas fraction) and the use of redox systems for detecting the presence of oxygen or the recording of colour changes which takes place in haemoglobin when it reacts with oxygen.

Each one of these conventional methods has at least one of the following disadvantages: high cost of apparatus, susceptibility of the apparatus to break-down or malfunctioning, difficulty of using the apparatus, long time required for determination, large sample volumes required, lack of specificity for the gas under investigation, measurement either only in the gaseous phase or only in the liquid phase or the necessity of using numerous and expensive chemicals.

It is an object of the present invention to provide a method which may be adapted to the individual purpose and is suitable for both gas and liquid samples and is employed in the same manner for two different gases and in which only inexpensive chemicals need to be replaced. At the same time, it is intended to shorten the time required for determination and to increase the sensitivity of the method.

In a first embodiment, the present invention provides a method for the determination of the oxygen or carbon dioxide gas content of a gaseous liquid sample which comprises injecting th'e sample from an injection syringe 21 into an injection chamber which is filled with an inert liquid and through which flows a continuous stream of a highly purified inert gas, continuously supplying a mixture of the gas from the gaseous or liquid sample and the inert gas and mixing the mixture with a reaction solution which undergoes a characteristic colour change in the presence of the gas, to be determined, while maintaining a constant rate of supply and constant proportion of sample to reaction solution in the mixture, and determining the concentration of the 2 or CO2 gas by measuring the degree of light absorption using a photometer.

In a second embodiment, the present invention provides a method for the determination of the oxygen or carbon dioxide gas content of a gaseous or liquid sample which comprises injecting the sample from a precision injection syringe through a gastight membrane into a transparent test cuvette which is closed on all sides and partly filled with a reaction solution which undergoes a characteristic colour change in the presence of the gas to be determined, thefremaining volume being occupied by an inert gas, and determining the concentration of the Oi, or CO2 gas by measuring the degree of light absorption using a photometer, the change in the degree of absorption being determined by comparison with a standard cuvette which is filled with the same quantity of solvent, but without the reagents.

The determination of oxygen is carried out by using a reaction solution which yields an intensive colouration with oxygen, preferably by using one or more hydroxy- or amino-derivatives of aromatic compounds, e.g. benzene or naphthalene, or derivatives of polycondensed heteroaromatic systems.

One or more colour intensifying substances, preferably metal salts, may be added, e.g.

ferrous ammonium sulphate (Mohr's salt).

The effective components are the Fe2+ ions in this case.

For determining the oxygen concentration, it has been found particularly suitable to use a reaction solution containing from 5 to 100 mmol/litre of pyrocatechol, from 1 to 20 mmol/litre of ferrous ammonium sulphate (Mohr's salt) and from 002 to 5 N sodium hydroxide solution.

The photometric determination of the reaction products is carried out in the system pyrocatechol + Fe2+ at a wavelength of approx. 490 nm, while in the case of pyrogallol as reactant a wavelength of approx.

620 nm is preferred.

The reaction mixture used for determining carbon dioxide is preferably a solution of hydrazine hydrate having a concentration of from 1 to 10 mmol/litre and fuchsin having a concentration of from 0105 to 0 5 mmol/litre. The photometric wavelength is approximately 545 nm in this case.

In the first embodiment, there may be used a pump of known type in which the sample of gas or liquid together with one or more reaction solutions is continuously pumped to the place where it is to be mixed while the components are maintained in a constant proportion to each other, and the mixture is transported to the photometer after the separation of gas bubbles. When transporting a liquid sample in this way, it is advantageous to introduce bubbles of an inert gas, in particular highly purified nitrogen, continuously into the stream of reaction mixture. Furthermore, before introducing a gas sample into this method, a zero adjustment should be carried out in the photometer by passing an inert gas, in particular highly purified nitrogen, into the stream flowing through the delivery channel and through the photometer.

Equalisation of pressure in the containers from which the reaction solutions are delivered may be established by washing out with a stream of an inert gas, in particular nitrogen, which has previously been passed through an alkeline pyrogallol solution.

Also in the first embodiment an injection chamber may be used which is traversed by a stream of inert gas entering from the underside through a gas-preamble and liquid-impermeable barrier, in particular a frit. This injection chamber is advantageously connected to a liquid-filled reservoir by means of an connecting pipe equip -ped with a shut-off valve.

The composition of the liquid depends on the type of the sample to be investigated and on the type of the gas. For the investigation of blood, potassium hexacyanoferrate (III) is used for expelling the oxygen, while carbon dioxide is expelled by diluted acetic acid.

The quantitative proportion of the gas to be investigated is calculated by planimetric measurements of the area under the graph in the curve tracer of the photometer or by means of an electronic integrator.

In the second embodiment, cuvettes which are sealed with a membrane made of a material which is gas-tight, but may be perforated are filled with the reaction solution used in the particular test.

Oxygen concentrations are measured by means of a test cuvette sealed by a membrane which is impervious to oxygen, but may be perforated. This cuvette is partly filled with inert gas and partly filled with a reaction solution which undergoes a colour change in the presence of oxygen.

This cuvette is made of a gas-impermeable material, preferably 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 10 mm (corresponding to a layer thickness of about 10 mm), external diameter 12 mm, capacity 3 to 5 ml.

The cuvette described above is sealed by a membrane which is impervious to oxygen, but may be perforated. The membrane is composed of several layers of an elastic support material, such as rubber, synthetic high molecular weight polvmers and gastight metal foils glued together, for example in the following arrangement: svnthetic high molecular weight polymer-metal (e.g.

aluminium) foil-rubber-metal (e.g. aluminium) foil-synthetic high molecular weight polymer. The cuvette is sealed by this membrane in a suitable manner, for example by glueing the membrane to it or by fixing it using a metal ring.

The sample of which the oxygen content is to be determined is injected from an in jection syringe, especially a -precision syringe, into the cuvette by perforating the membrane. - After removal of the syringe, the elastic foils of the membrane ensure 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.

The oxygen concentration measurement described is extremely simple to carry out.

Since -all that is required is to inject a from 10 to 100 !all sample from a precision syringe into the cuvette and to read off the absorp tion -change in a photometer, this test may be carried out by unskilled operators. Moreover, measurement of the oxygen concentration takes very little time, at the most 1 minute from introducing the blood sample to obtaining the result.

Due to the presence of an alkaline medium in the reaction solution mixture, any cells present in samples of biological material (for example erythrocytes in whole blood samples for oxygen determination) undergo lysis and the cell membranes dissolve.

Other organic corpuscular constituents, for example those found in river water (e.g.

algae) are also dissolved. If desired, this process of solution may be facilitated by the addition of detergents, e.g. from 1 to 3%, by weight, 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, may take place in the reaction solution mixture used for the colour reaction, the measurements of oxygen concentration may also be carried out in those biological systems in which the oxygen concentration would be greatly altered by biological processes if measurements took too long so that no significant results could be obtained.

By diluting the sample (from 10 to 100 1) to the volume of the reaction solution mixture, i.e. to about 2 5 ml, even intensely coloured samples (e.g. blood) may 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 may 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, may be dispensed with. The equalisation of pressure which is necessary when injecting liquid samples may be achieved by the presence of from 1 to 2 ml of inert gas in the cuvette, corresponding to from 30 to 40% of the total volume.

Measurement of the oxygen concentration may be carried out in a mobile unit if a portable, battery operated photometer is used for the absorption measurement.

Diagnosis of the oxygen concentration of human blood may therefore be carried out on the spot, for example, in emergency and accident cases (e.g. medical ambulances), in diving, air travel and space travel medicine (e.g. submarines or aircraft) and in sports medicine and industrial medicine.

The first embodiment illustrated in accompanying Figure 1 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 containing 9 liquid. A stream of highly purified - nitrogen entering the injection chamber from below through a fruit bottom plate 23 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 a precision pump.

This pump continuously delivers the reaction solutions in constant proportions into the mixing spiral from which the coloured solution is transferred to a photometer. The liquid in the injection chamber may be replaced by liquid from a reservoir 26 by opening the tap 25 in a connecting pipe 24 or it may be run off through a discharge pipe 27 by opening the tap 28.

The second embodiment merely requires a precision injection syringe, a test cuvette closed on all sides and capable of being perforated on one side, which cuvette contains one of the various reaction solutions, and a photometer into which the test cuvette is inserted after injection of the sample shaking and a short waiting period (from 20 to 30 sec.), and in which the cuvette is inspected. The change in the degree of absorption is obtained by comparing the cuvette with a standard in the form of a cuvette which is filled with the same quantity of liquid, but without the reagents used for the test.

This process thus does not operate continuously with the aid of a precision pump, but intermittently. An example of a test cuvette for measuring oxygen concentrations is shown in accompanying Figure 2.

This is a cylindrical glass cuvette 31, in which the length of the light path is 10 mm.

The cuvette contains 2-5 ml of reaction liquid 32; the remaining space in the cuvette is filled with inert gas 33. The cuvette 31 is closed by a membrane 34 composed of several layers. The membrane is pressed against the top surface of the cuvette bv a screw fitting 35. The sample is injected through the membrane from a injection svringe. The membrane (accomDanving Figure 2d) is composed of the following layers: polyethylene 36, aluminium 37, Para rubber 38, aluminium 39, polyethylene 310.

The advantages of the methods described above should be obvious. They enable the same apparatus to be used for determining two different gases, simply by replacing reagents. The methods are so simple that they may be carried out by unskilled operators. The high sensitivities also play an important part. For the first-described method the lower limit of detection is 01 microlitre for oxygen and 1 microlitre for carbon dioxide (under standard physical conditions), using a relatively simple photometer. The second method is particularly suitable when only small quantities of sample are available and may be used for gas or liquid samples measuring from 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 light path in the cuvette may be 10 mm so that the cuvette may be used in a holder of a conventional photometer.

For medical diagnostic purposes, this means that venous punctures are not required for obtaining the blood sample (medical ancillaries may take the necessary blood sample from the ear lobe), or that oxygen determination may be carried out even when only small quantities of blood are available (determination of the oxygen concentration in the blood taken from the scalp in infants).

In contrast to other methods, this method does not measure the oxygen partial pressure, but the oxygen concentration. This is of considerable diagnostic importance for numerous cases, for example in poison cases in which methaemoglobin is formed or in carbon monoxide poisoning.

Another noteworthy feature of the method is the simplicity of the apparatus required, which is reflected in low initial costs, as well as in low running costs, since only inexpensive chemicals are used. This simplicity in the apparatus also makes it very mobile so that the second method, in particular, may also be used in mobile field units for environmental monitoring and control operations.

Other important factors are the high degree of variability and adaptability to the given purpose resulting from the fact that methods are available which cover all the possibilities of gaseous or liquid samples, large or small quantities of samples and two different types of gas.

In all methods, moreover, the analysis time is reduced to 2 minutes.

It should be noted that it is not possible to carry out a simultaneous determination of 0, and CO2. If it is desired to estimate both gases, separate determinations must be carried out.

WHAT WE CLAIM IS: - 1. A method for the determination of the oxygen - or carbon dioxide gas content of a gaseous or liquid sample which comprises injecting the sample from an injection syringe into an injection chamber which is filled with an inert liquid and through which flows a continuous stream of a highly purified inert gas, continuously supplying a mixture of the gas from the gaseous or liquid sample and the inert gas and mixing the mixture with a reaction solution which undergoes a characteristic colour change in the presence of the gas to be determined while maintaining a constant rate of supply and constant proportion of sample to reaction solution in the mixture, and determining the concentration of the 02 or CO-2 gas by measuring the degree of light absorption using a photometer.

2. A method for the determination of the oxygen or carbon dioxide gas content of a gaseous or liquid sample which comprises injecting the sample from a precision injection syringe through a gas-tight membrane into a transparent test cuvette which is closed on all sides and partly filled with a reaction solution which undergoes a characteristic colour change in the presence of the gas to be determined, the remaining volume being occupied by an inert gas, and determining the concentration of the 0, or -CO2 gas by measuring the degree of light absorption using a photometer, the change in the degree of absorption being determined by comparison with a standard cuvette which is filled with the same quantity of solvent, but without the reagents.

3. A method as claimed in Claim 1 in which the inert liquid is distilled water.

4. A method as claimed in Claim 1 or Claim 2 in which the inert gas is nitrogen.

5. A method as claimed in any of Claims 1 to 4 in which the reaction solution which undergoes a colour change in the presence of oxygen comprises one or more hydroxyl- or amino-derivatives of aromatic compounds or of derivatives of multinuclear aromatic compounds containing one or more heteroatoms and optionally one or more colour intensifying metal salts.

6. A process as claimed in Claim 5 in which the reaction solution comprises 25 mM of pyrocatechol in 1 N sodium hydroxide or potassium hydroxide.

7. A method as claimed in Claim 5 or Claim 6 in which the metal salt is 5 mM of Fe2e in the form of Fe(NH4)2(SO4),.

8. A method as claimed in Claim 1 in which a pump continuously delivers the gas or liquid sample together with one or more reaction solutions to the place where the components are mixed and, after separation of gas bubble, to the photometer.

9. A method as claimed in Claim 8 in which bubbles of an inert gas are contains;

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. rubber 38, aluminium 39, polyethylene 310. The advantages of the methods described above should be obvious. They enable the same apparatus to be used for determining two different gases, simply by replacing reagents. The methods are so simple that they may be carried out by unskilled operators. The high sensitivities also play an important part. For the first-described method the lower limit of detection is 01 microlitre for oxygen and 1 microlitre for carbon dioxide (under standard physical conditions), using a relatively simple photometer. The second method is particularly suitable when only small quantities of sample are available and may be used for gas or liquid samples measuring from 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 light path in the cuvette may be 10 mm so that the cuvette may be used in a holder of a conventional photometer. For medical diagnostic purposes, this means that venous punctures are not required for obtaining the blood sample (medical ancillaries may take the necessary blood sample from the ear lobe), or that oxygen determination may be carried out even when only small quantities of blood are available (determination of the oxygen concentration in the blood taken from the scalp in infants). In contrast to other methods, this method does not measure the oxygen partial pressure, but the oxygen concentration. This is of considerable diagnostic importance for numerous cases, for example in poison cases in which methaemoglobin is formed or in carbon monoxide poisoning. Another noteworthy feature of the method is the simplicity of the apparatus required, which is reflected in low initial costs, as well as in low running costs, since only inexpensive chemicals are used. This simplicity in the apparatus also makes it very mobile so that the second method, in particular, may also be used in mobile field units for environmental monitoring and control operations. Other important factors are the high degree of variability and adaptability to the given purpose resulting from the fact that methods are available which cover all the possibilities of gaseous or liquid samples, large or small quantities of samples and two different types of gas. In all methods, moreover, the analysis time is reduced to 2 minutes. It should be noted that it is not possible to carry out a simultaneous determination of 0, and CO2. If it is desired to estimate both gases, separate determinations must be carried out. WHAT WE CLAIM IS: -

1. A method for the determination of the oxygen - or carbon dioxide gas content of a gaseous or liquid sample which comprises injecting the sample from an injection syringe into an injection chamber which is filled with an inert liquid and through which flows a continuous stream of a highly purified inert gas, continuously supplying a mixture of the gas from the gaseous or liquid sample and the inert gas and mixing the mixture with a reaction solution which undergoes a characteristic colour change in the presence of the gas to be determined while maintaining a constant rate of supply and constant proportion of sample to reaction solution in the mixture, and determining the concentration of the 02 or CO-2 gas by measuring the degree of light absorption using a photometer.

3. A method as claimed in Claim 1 in which the inert liquid is distilled water.

ously introduced into the stream delivering a liquid sample.

10. A method as claimed in Claim 8 or Claim 9 in which, before a gas sample is introduced into the delivery stream, a zero determination is carried out by passing an inert gas through the channel carrying the delivery stream and through the photometer.

11. A method as claimed in Claim 9 or Claim 10 in which the reaction solution(s) is/are delivered from one or more containers in which pressure equalisation is established by a following stream of inert gas.

12. A method as claimed in Claim 11 in which the inert gas is nitrogen which has previously been passed through an alkaline pyrogallol solution.

13. A method as claimed in Claim 1 in which the inert gas enters the injection chamber from the underside through a barrier which is permeable to gas and impermeable to liquid.

14. A method as claimed in Claim 1 in which the injection chamber is connected to a liquid-filled storage vessel through a connecting pipe equipped with a shut-off valve.

15. A method as claimed in Claim 1 in which a liquid which accelerates the release of the gas which is to be determined from a liquid sample is used.

16. A method as claimed in Claim 1 in which the quantitative proportion of the gas to be determined is calculated by planimetric measurements of the area under the curve in a curve tracer of the photometer or by means of an electronic integrator.

17. A method as claimed in any of Claims 1 to 16 in which the photometer uses light having a wave-length of approx.

490 nm or approx. 520 nm while pyrocatechol or pyrogallol is used for oxygen determination.

18. A method as claimed in any of Claims 1 to 16 in which the photometer uses light having a wavelength of approx.

545 nm for carbon dioxide determination.

19. A method as claimed in any of Claims 1 to 18 substantially as herein described.

20. A method as claimed in any of Claims 1 to 18 substantially as herein described with reference to the accompanying drawings. 1 -- -