GB1588761A - Reference liquid for blood gas equipment - Google Patents

Description

(54) A REFERENCE LIQUID FOR BLOOD GAS EQUIPMENT (71) We, RADIOMETER A/S, a Danish Limited Company, residing at Emdrupvej 72, DK-2400 Copenhagen NV, Denmark, 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:- The present invention relates to a reference liquid for quality control and/or calibration of blood gas measuring equipment.

Blood gas measuring apparatuses are known which are designed for measuring, by means of suitable measuring electrodes, blood pH, concentration of dissolved carbon dioxide in blood, expressed as Pco2 (the partial pressure of carbon dioxide) and the concentration of dissolved oxygen in the blood, expressed as Po2 (the partial pressure of oxygen), and one known fully automatic blood gas measuring apparatus (Radiometer ABLI, described, e.g. in U.S. patent No.

3,874,850) also simultaneously measures the blood hemoglobin content (Hb) which is otherwise normally measured separately.

From these four central parameters may be calculated various derived parameters which are of great significance to the judgement of so-called acid-base status of the organism.

The measurements referred to above are all relative measurements where the unknown sample is compared with standards. Hence, the quality of these standards is decisive to the quality of the measurement of the single parameters.

When using manual or semiautomatic blood gas measuring equipment, great technical skill is nowadays a requirement to the user of the measuring equipment in order to obtain measurements of satisfactory quality. The technical level of the user may be lower when fully automatic self-calibrating equipment is used, e.g. of the type described in the above-mentioned U.S. patent, but this does not remove the necessity or desirability of being able to check the measuring quality of the equipment, including the quality of the standards, calibration liquids, etc., of the equipment, using a known reference.

Even though it is, in principle, generally known to check a measuring equipment by introducing a sample of known properties into the equipment, this is a great problem in connection with equipment for measuring pH-Pc02-P02- and optionally Hb.

A sample (a blood sample or another aqueous solution) of this type is normally not stable during any longer period (CO2 and O2 escape from the sample) which means that the sample must be prepared on the spot by the user. Normally, this gives rise to problems involving extraordinarily much labor, expensive extra equipment and uncertainty, as the preparation process is technically rather complicated.

All over the world, there is in our days an interest in a control system for measuring values from equipment of the type mentioned, as this equipment is used directly in connection with patient treatment and often under extremely critical circumstances (e.g. during surgery).

In the U.S.A., Congress has dealt with this problem during recent years, and at present, the legislation tends toward requiring that the "supplier of blood data", e.g. the head doctor of the laboratory, shall be able to prove, at any time, that the measuring equipment used is able to yield reliable data in that it has been checked by means of a system independent of the normal calibration system of the equipment (quality control).

Hence, the general desire to-day (also outside the U.S.A.) is that one would be able to buy for this quality control, small containers with samples of known composition and of great reliability.

All blood gas measuring equipment commercially available requires frequent calibration, usually with intervals of some hours. For this purpose, the known art uses, for certain types of equipment, various calibration liquids, some of which (e.g. pH buffer mixtures) are commercially available in small containers and show high reliability with respect to keeping the stated pH values, whereas the calibration liquids for the calibration of other parts of the measuring equipment, e.g. the Pco2 measuring equipment and the Po2 measuring equipment, are at the moment not commercially available in suitable containers and with well-defined data, but will have to be prepared on the spot, which leads to the above problems. Some technically advanced blood gas measuring equipment, e.g. the above-mentioned fully automatic Radiometer ABLI blood gas measuring equipment, use solutions which in the equipment proper are equilibrated with known gas mixtures to obtain well-defined values for pH, Pro2, and Po2, and the calibration liquids thus prepared in the equipment and showing well-defined data are used for the calibration within the equipment, without being transferred to separate containers.

It would be of high interest to be able to perform the very calibration of the blood gas measuring equipment, especially semiautomatic equipment, using a handy reference liquid which may be produced and packed in suitable unit portions and distributed and stored with retention of its relevant data with high exactitude and reliability, in order that the calibration of the blood gas measuring equipment can be performed simply by introducing a unit portion or a part thereof in the equipment without the necessity of any special preparation or checking of the liquid.

The present invention provides a reference liquid which may be used for quality control and/or calibration of blood gas measuring equipment, and which is known with respect to the parameters pH, Pco2, Po2, and, if desired, a parameter representing the hemoglobin concentration.

U.S. patent specification No. 3,681,255 mentions the possibility of using a single calibration liquid with known hydrogen carbonate ion concentration, known partial pressure of carbon dioxide (and consequently in accordance with the known Henderson-Hasselbalch equation, known pH) and known partial pressure of oxygen for calibration of the measuring electrodes in blood gas measuring equipment, and according to the above-mentioned U.S. patent specification, said calibration liquid may be delivered to the user in a gas-tight container.

Although calibration liquids of this type would, due to the inclusion in a gastight container with retention of the known parameters, in principle be suitable for quality control and calibration of blood gas measuring equipment, it is difficult in practice, using the known calibration liquids, in other words in the actual performance of the quality control or calibration by introducing the reference liquid into the blood gas measuring equipment, to fulfil such exactitude requirements which may reasonably be made on a reference liquid for calibration and quality control. Especially, the Po2 control involves serious difficulties, and with the known art calibration liquids contained in gas-tightly sealed containers, it is not possible to obtain any reliable control or calibration of the Po2 system under all circumstances.

The reference liquid according to the present invention is contained in a gastight container and shows, at a fixed temperature, a known pH; a known partial pressure of carbon dioxide and a known partial pressure of oxygen; and additionally may contain oxygen reversibly contained in a dispersed organic substance which is able to contain, per volume unit, a larger amount of oxygen than water can contain; and optionally a coloring component.

The reference liquid according to the invention may contain a coloring component and then may be used for quality control and/or calibration of the equipment with respect to the determination of the amount of hemoglobin, in addition to the quality control and/or calibration of the equipment with respect to the determination of pH, Pco2 and Po2.

According to the present invention there is provided a synthetic reference liquid for quality control and/or calibration of blood gas measuring equipment, said reference liquid showing, at a fixed temperature, a known pH, Pco2, and Po2 value, said reference liquid comprising an aqueous phase containing at least one pH buffer system and particles suspended in the said aqueous phase, the particles being either 1) polymer-coated microcapsules containing at least one organic substance which shows a greater ability to reversibly contain oxygen than does water, or 2) porous particles of a solid substance.

The term "reversibly contained" is intended to describe that the oxygen is present in the organic substance in such a manner that the organic substance is able to deliver or take up oxygen under the manipulation and measuring conditions, so that the organic substance will, due to its ability to contain larger amounts of oxygen per volume unit than water, increase the oxygen buffer capacity of the reference liquid. The term "reversibly contained may comprise both such cases in which the oxygen is dissolved or otherwise predominantly physically bound in the organic substance, and such cases where the oxygen is predominantly chemically bound, especially complex bound, to or in the organic substance in question.

As organic substances in which the oxygen is dissolved or otherwise predominantly physically bound may be used water soluble organic compounds which do not adversely influence the remaining parameters in the reference liquid and which show substantially greater ability to dissolve oxygen than does water, It is known that the solubility of oxygen in various water soluble organic compounds, e.g. alcohols such as ethanol, propanol, and tert.butanol, is considerably greater than the solubility of oxygen in water, and in an aqueous solution of such organic compounds, in other words for example an aqueous solution of one of the alcohols mentioned, the solubility of oxygen and, consequently, the oxygen buffer capacity is considerably greater than in an aqueous solution without such dissolved organic components. However, as the increase of oxygen solubility which is desirable to obtain a reliable reference liquid for the calibration or control of the Po2 system is very large, preferably about 1025 times the solubility in water and at least about 5 times, and as so large increases in solubility are difficult to obtain with dissolved organic substances, their use alone does not constitute a preferred embodiment of the present invention.

When the reference liquid according to the invention contains, as oxygen buffer capacity increasing organic substance, a dissolved organic substance in which oxygen is considerably more soluble than in water, the concentration of this dissolved substance is usually lO60V0, especially 20-400/,, of the total volume.

The solubility of oxygen in various organic materials which cannot be considered substantially water soluble, is even greater than the solubility of oxygen in the above-mentioned water soluble organic materials. Such non-water soluble organic materials showing a great soluting power for oxygen, are e.g. oils or oily synthetic organic substances and organic polymers. As examples may be mentioned hydrocarbons such as isooctane, silicone oils and silicone rubbers and fluorocarbon compounds, i.e. fluorinated, especially perfluorinated, hydrocarbons and compounds containing such fluorinated hydrocarbon groups, as well as polymers thereof. With such non-water soluble organic materials which are liquids of lipoid character or solid substances, dispersions may be prepared which are emulsions of lipoid-in-water-type or suspensions. For the purpose of the present invention, such systems show the advantage that the great oxygen-soluting power of the lipoids or solids results in a large oxygen buffer capacity, whereas the liquid still retains its property as aqueous solution and hence, continuously permits the establishment of a PcoJpH buffer system. Hence, especially preferred reference liquids according to the invention contain oxygen dissolved in emulsified or suspended non-water soluble organic substances. Of course, it is also possible to use combinations of water soluble and non-water soluble organic substances with large capacity for taking up oxygen.

Quite generally,the following criteria should apply to the selection of the nonwater soluble organic substance used as an oxygen carrier: 1. The oxygen carrier should have high soluting power for oxygen (reversible).

2. The oxygen carrier should show no (or little) protolytical activity.

3. The organic phase consisting of the oxygen carrier should be dispersible in an aqueous buffer to fulfil the following requirements: a) High concentration of organic phase (which give a high capacity of the mixture). b) The mixture should be stable. c) The density of the mixture should be less than or equal to the density of blood, on account of blood gas measuring equipment containing salt bridges, the use of which is dependent on the blood having a smaller density than the salt solution. d) The viscosity of the mixture should not be too high.

4. The oxygen carrier should be extensively chemically inert (in order to not destroy the structural materials in the blood gas measuring equipment).

5. The oxygen carrier should preferably be undangerous to handle, and 6. The oxygen carrier should preferably be a commercially available material and, hence, cheap and easy to obtain.

As an illustration of the increase in solubility for oxygen which is obtained by using one of the substances mentioned, water soluble or non-water soluble, it may be mentioned that the solubility of O2 (at 1 atmosphere's oxygen pressure and 25"C) in water is 2.4 /" v/v, while it is 21% v/v in ethanol, 12% v/v in olive oil, typically 50% v/v in fluorocarbon compounds, typically 18% v/v in silicone rubbers, typically 20 /" v/v in silicone oils, and about 36% v/v in isooctane.

The amount of dissolved oxygen in various systems as function of the partial pressure of oxygen (single logarithmical plot) is shown in Fig. 1 in which "fluorocarbon" designates perfluorotributylamine. It will be noted that while even a very small change in the oxygen amount in pure water will result in a very large difference in Po2, a change of the oxygen amount in, e.g., the fluorocarbon compound will result in a far smaller change in Po2, and that, e.g., a 40% emulsion of the fluorocarbon compound in water shows a far better oxygen buffer capacity than water, i.e., shows far less Po2 change at a given change in oxygen content.

It is preferred that the suspended phase constitutes at the most 80 /" of the total volume, especially 4070%, as the water phase should of course be of a sufficient proportion so as to avoid a decrease in the quality of the pH measurement. Hence, it is clear that in composing the liquid according to the invention, one will preferably choose such suspended components which show an especially high solubility for oxygen, e.g. the above-mentioned fluorocarbon compounds.

As examples of fluorohydrocarbons and fluorohydrocarbon group-containing compounds, in other words fluorocarbon compounds, which are useful for the purpose of the present invention may be mentioned perfluorotributyl amine ((C4F9)2N), which is sold by 3M Company under the designation "FC 43", perfluoromethyl cyclohexane and perfluorodimethyl decaline.

Because of its good ability to dissolve oxygen, perfluorotributyl amine is a preferred compound. As an example of a silicone rubber useful as suspended phase in the reference liquid according to the invention may be mentioned silicone rubber CAF4/60 Rhodorsil (Registered Trade Mark), Rhone Poulenc, Paris.

According to a particular aspect of the present invention, a suspended solid is used which is porous and which may, in its porosities, contain oxygen or an oxygencontaining gas, and a suitable material for this purpose is porous silicone rubber.

Porous silicone rubber useful for this purpose may, for example, be prepared by applying, prior to and/or during the curing of the silicone rubber, a superatmospheric pressure which is thereafter released during the curing. Porous silicone rubber useful for this purpose may also be prepared by dispersing in the silicone rubber, prior to the curing, a volatile non-solvent for silicone rubber, for example methanol, and thereafter curing the silicone rubber and evaporating the volatile substance.

To obtain a stable suspension it may be necessary that the reference liquid according to the invention contains a suitable suspending agent, and this agent may be of any type which does not adversely influence the parameters to be determined by means of the reference liquid, and which gives a stable suspension of the organic material selected. Suitable suspending agents for this purpose are commercially available.

As mentioned above, the organic substance in which the oxygen in the reference liquid according to the invention is reversibly contained may be a substance to which oxygen is reversibly chemically bound, especially complexbound.

Most chemical processes involving oxygen are characteristic in that the processes are substantially irreversible, so that an oxygen-containing compound once formed will not to any substantial degree be able to liberate oxygen or, expressed in another manner, the oxygen-containing compounds formed by the irreversible processes are not suitable fot increasing the oxygen buffer capacity of the reference liquids according to the present invention.

However, reversible oxygen processes do exist, e.g. in the blood hemoglobin molecule, which is able to reversibly take up and give off oxygen in substantial amounts, and which therefore in principle would be excellently suitable for the purpose of the present invention. However, outside the organism, the hemoglobin molecule, like other biological substances such as proteins or proteinaceous complexes, is relatively unstable, and reference liquids prepared with hemoglobin or other biological substances like proteins or proteinaceous complexes as oxygen capacity-increasing organic substance will, therefore, show the disadvantage that they are stable only for relatively short periods unless special precautions are taken to secure their stability, e.g. freezing of the reference liquid immediately or shortly after its preparation and packing, distribution and storing of the reference liquid in frozen form, addition of suitable stabilizers to prevent chemical degradation and suitable sterilization or addition of antibiotics to prevent microbial degradation.

However, other-and simpler and less sensible-organic compounds than the hemoglobin molecule are known which are able to reversibly complex-bind oxygen. As examples of such non-biological compounds may especially be mentioned metal organic compounds of transition group metals, especially cobalt or iron, in which the metal is bound, usually complex-bound, to nitrogencontaining groups, e.g. transition group metal complexes with porfyrine-like compounds such as iron(II)phthalocyanine tetrasulfonic acid.

For the purpose of the present invention, the organic substance which is able to chemically bind, especially complex-bind, oxygen reversibly should preferably be one which has a suitable position of the equilibrium of the reversible oxygen reaction in question, i.e. a position of the equilibrium which resembles that of hemoglobin (the greatest resemblance with authentic blood), and/or a position of the equilibrium which results in an optimum oxygen buffer capacity at or about the Po2 value which is to be possessed by the reference liquid.

With respect to the position of the equilibrium of the oxygen reaction of hemoglobin, the following applies when the oxygen uptake of the blood is considered in a simplified manner: Hb+O2zHho2 wherein Hb is the hemoglobin molecule, O2 is the oxygen molecule, and HbO2 is the oxygen-containing hemoglobin complex.

The solubility of oxygen (in free form) in the water phase of blood can reasonably be put at 1.4 10-6 mole of oxygen per liter per mm Hg oxygen partial pressure. Empirically, at an oxygen partial pressure of 27 mm Hg, equal amounts of on the one hand hemoglobin on the Hb form and on the other hand hemoglobin on HbO2 form are present in the blood. At this partial pressure, the concentration of dissolved oxygen in the water phase of blood is: [021=1.4 10-6. 27~3.8 10-5 The stability constant K for the oxygen-containing hemoglobin complex is [HbO2] K= [Hb] [O2] and as (vide above) [HbO2]=[Hb] at [O2]#3.8#10-5, the following applies: 1 K= #K=104.5 3.8 10-5 From this follows that among the oxygen complex-forming organic compounds binding oxygen in the same manner as hemoglobin, i.e. according to the above reaction scheme, the ideal compounds for use as oxygen buffers in a reference liquid which is very close to the properties of blood in this regard will be such compounds, for which the stability constant of their oxygen complex is about 04'5, e.g. in the range of 1031055, especially 104--105.

Another type of the (different from hemoglobin) organic compounds which reversibly form complex with oxygen, bind oxygen according to the reaction scheme: 2L+O2L-O2-L wherein L is the ligand which is able to bind oxygen, and L-O2-L is the complex compound in its oxygenated form.

The stability constant of the above-mentioned oxygenated complex compound L-O2-L is [L-O2-Ll K= [L]2[O2] wherein [ 2] is the concentration of oxygen (in free form) dissolved in the system in question. Like the above calculated stability constant for the oxygenated hemoglobin complex, this constant is, to some extent, temperature dependent; however, for the purpose of the present invention, this temperature dependency can usually be disregarded. When the concentration of L is designated α, and the concentration of L-O2-L is designated , α+2 =c (cf. the reaction scheme), or α=c-2 , c being the total concentration of the ligand. From this follows:

=K= α2[O2] (c-2 )2[O2] =K(c-2 )2[O2] (c-2 )2=c2+4 2-4c =K[O2]2+4K[O2] 2-4K[O2]c 4K[O2] 2-4K[O2]c - +K[O2]c2=0 4K[O2] 2-(4K[O2]c+1) +K[O2]c2=0 (4K[O2]c+1)#((4K[O2]c+1)2-16K[O2]K[O2]c2) = 8K[02] wherein , as appears from the above, represents the concentration of complexbound oxygen.

The total oxygen concentration in complex systems, TO2, is TO2=[O2]+ wherein [ 2] is the concentration of dissolved oxygen, and A is the concentration of complex-bound oxygen.

Decisive to the suitability of the oxygen complex-forming compound for use in the liquid according to the invention is that around the oxygen partial pressure which the reference liquid is to possess, a suitable oxygen buffer capacity should be obtained, which means that any loss of small amounts of oxygen from the liquid, or any gain of small amounts of oxygen to the liquid, e.g. during the manipulation of the liquid and during a calibration operation, should result in as small a change in the Po2 of the liquid as possible. A high oxygen buffer capacity is of course in principle obtained when the concentration of the oxygen complex-forming compound in the reference liquid is high, but the oxygen partial pressure around which the buffer effect has its optimum depends on both the concentration mentioned and on the size of the above-mentioned stability constant K. When composing a reference liquid according to the invention using an oxygen complexforming compound, one should, therefore, as oxygen complex-forming compound preferably select such compound in such concentration that optimum oxygen buffer capacity is obtained around the oxygen partial pressure to be possessed by the reference liquid. In practice, the oxygen buffer capacity for the abovementioned complex system can be defined as d d log [O2] wherein p and [ 2] are as defined above, and hence, the maximum oxygen buffer capacity is the one at which d2 =O, d(log[O2])2 which for this function is synonymous with d2 log [O2] = O. d 2 As = K it follows that [O2] = (c - 2 )2[O]2 K(c - 2 )2 log 102] = -log K + log - 2 log (c - 2 ) d log (02] d (c - 2 ) d log 102) d (c - 2 ) d d d log [O2] 1 1 = - (log e) - 2 log e (- 2) d c - 2 d log [O2] 4 = log e (1/ + ) , d d2 log [O2] - log e = - + log e (-4(c - 2 )-2 (- 2)) d 2 2 d2 log [O2] = log e d

From this follows that

8 = 1/ 2 (c - 2 )2 8 2 = (c - 2 )2 = c2 + 4 2 - 4c 4 2 + 4 c - c2 = o

On this basis, there may be calculated, as examples, suitable stability constants for oxygen complexes for use in reference liquids, the oxygen partial pressure of which is to be at one of the three values at which it is often desired to calibrate, i.e.

500 mm Hg, 150 mm Hg, and 50 mm Hg. For these, the following is true: 1) [O2]=6#10-4 mol/liter (#500 mm Hg) 2) [O2]=2 10-4 mol/liter (#150 mm Hg) 3) [O2]=6 10-5 mol/liter (#50 mm Hg) If it is desired to use the ligand in these cases in a concentration of 10-1 mol/liter, the following values are calculated for the stability constant K which at the three [O2] values mentioned gives maximum oxygen buffer capacity:

K= (cf. above) [c-2 ]2[O2] 0.207. c 0.207. c K= = (c-2 0.207 c)2[O21 (0.586 c)2[O2] 0.603 K c#[O2] 0.603 K1= =104 10-' 6 10-4 0.603 K2= =3 x 104 10-1#2#10-4 0.603 K3= =105 10-1#6#10-5 Fig. 2 shows, as a single logarithmical plot, the concentration of total oxygen amount as function of the partial pressure of oxygen for oxygen complexes with the three above-mentioned stability constants, each of them in a concentration of 10-' mol/liter in the reference liquid. It will be seen how the oxygen complex compounds show a considerable oxygen buffer capacity (considerably greater steepness of the graph than for H2O) in a broad range around the partial pressures mentioned, so that loss or gain of a certain amount of oxygen in the range stated will result in per cent-wise the same (relatively small) change in the oxygen partial pressure of the system.

On the background of the examples given above and Fig. 2 it can be stated that also for complex compounds binding oxygen according to the reaction scheme 2L+O2#L-O2-L, beneficial stability constants will be in the range of 103-105.5, especially 104-105.

When the reference liquid according to the invention contains, as oxygen buffer capacity-increasing organic substance, an oxygen complex-forming compound, the concentration of this compound is preferably between 10-4 and I mol per liter, especially from 10-3 to 5 10-' mol per liter.

When the reference liquid according to the invention contains a coloring component which is to "simulate" hemoglobin and permit the use of the reference liquid for quality control and/or calibration of the hemoglobin measuring part of the blood measuring equipment, the coloring component is preferably one having an absorption maximum at or around an isobestic point of the system hemoglobin/hemoglobin-oxygen-complex (in other words, to use the symbols used above, Hb/HbO2), i.e. the point in which the molar extinction caused by Hb has the same size as the molar extinction caused by HbO2, as blood gas measuring equipment comprising a hemoglobin measuring part is usually equipped with such filters that the absorption of the sample introduced is measured at or in a narrow range around one of the isobestic points, e.g. the point at 505 nm. Hence, dyestuffs having absorption maximum around 500 nm are suitable for use in the reference liquid according to the invention. As examples of such dyestuffs may be mentioned Amaranth, Allura Red and Ponceau 4 R, 70%. The last mentioned dyestuff is a chemical azo dyestuff having C.I. No. 16,255 (1956). The compound is the trisodium salt of l-(4-sulfo-l-naphthyl-azo)-2-naphthol-6,8-disulphonic acid. The dyestuff used should suitably be contained in the liquid in such concentration that it corresponds to the extinction of human blood, which, for Ponceau 4 R, 70% means either by interface polymerization or by interface precipitation. As examples of materials which may be formed by interface polymerization may be mentioned proteins, polyamides (nylon), polyurethane, polyurea, polysulphonamide or polyphenyl esters, and as examples of polymer materials which may be precipitated in the interface may be mentioned cellulose nitrate, protein, polystyrene and "Silastic" (Registered Trade Mark) (which latter, however, requires a subsequent vulcanization). According to the invention it is preferred that the polymer coating of the microcapsules is a nonbiological and non-proteinaceous material, especially nylon formed in situ.

The size of the microcapsules is preferably of the order of 1--5, and the polymer coating is preferably of the order of 300-600 ngstrm.

By including the oxygen buffer capacity-increasing substance in a polymer coating in the form of microcapsules, there is obtained firstly a more stable suspension than through emulsification of the same substances, and secondly, there is obtained the advantage that oxygen buffer capacity-increasing substances which would otherwise present problems because of their aggressivity towards parts of the blood gas measuring apparatus, for example the polypropylene membranes of the oxygen electrodes, may be used in the reference liquid substantially without any problems. Furthermore, by using the microcapsules, it bacomes possible to combine several substances in the organic phase in such a way that its density becomes equal to or very close to the density of water which counteracts any tendency for the disperse phase to separate from the aqueous phase, and the combination of several substances can also contribute to improve other properties, for example the strength and smoothness of the polymer membrane around the microcapsules. Thus, for example, addition of carbon tetrachloride to isooctane encapsulated in nylon contributes to increase the smoothness of the nylon membranes and to reduce the tendency of the microparticles to agglomerate.

The preparation of a suspension of microcapsules of the above-described kind is performed by suspending at least one organic substance which shows considerably greater ability to reversibly contain oxygen than does water, in an aqueous phase under such conditions that the single suspended particles are coated with a polymer material. A technique which, in certain respects, resembles this process is described in the literature ("Semipermeable Aqueous Microcapsules", T. M. S. Chang, F. C. Machintosh, S. G. Mason, Can. J. of Physiology and Pharmacology 44, 115-128 (1966), "Artificial Cells", Thomas Ming Swi Chang, Charles C. Thomas, Springfield, Illinois, U.S.A. 1972). In these articles, methods are disclosed for the preparation of thin, stable polymer membranes around aqueous microdrops, either through interface polymerisation or through interface precipitation. The enclosed aqueous phase could contain enzymes and/or other proteins and was separated from a continuous aqueous phase by means of the semipermeable membranes formed. In the above-mentioned works, the two thus separated aqueous phases were to serve as a model for biological cell walls. By means of such a model, the study of the single cell functions were facilitated. This required a semipermeable membrane between two aqueous phases so that exchange of water, salts and small molecules could take place, whereas larger molecules (e.g. enzymes) were retained. The preparation of the polymer membranes around the aqueous microdrops in this known art was performed in the following manner: 1) The aqueous protein solution (possibly containing organic diamine) was emulsified in an organic liquid by means of a suitable soap.

2) By addition of a suitable material to the continuous phase, a polymer membrane was formed around each droplet. The membrane formation was performed either by interface polymerisation, in other words a chemical process, or by interface precipitation, in other words a physical process, dependent on the lower solubility for a polymer in the interface.

3) By means of suitable solvents and/or surface active agents, the microcapsules formed were removed from the organic phase and suspended in an aqueous medium. In a specific example, the already previously investigated interface polymerization (Morgan and Kwolek, J. Pol. Sci. 40, 299, 1959) between a water soluble aliphatic diamine and a solution of a dicarboxylic acid chloride in an organic solvent was utilized. Chang et al. showed that polymerisation also took place when the water phase was present finely divided in the organic phase.

In contrast to the above known art, the coating according to the present invention aims at a stabilisation of a disperse organic phase in an aqueous medium and a reduction of the diffusion rate of the liquid phases through the membrane, and a characteristic difference in comparison with the method according to Chang et al. is that in the process of this invention, an organic phase, rather than the water phase, is finely divided.

In preferred embodiments of the process according to this invention, the polymeric coating material is formed by interface polymerisation between polymer-forming components, one of which being present in the organic phase, and the other one being present in the aqueous phase. For example, in accordance with a preferred embodiment, nylon is formed in the interface in that the organic phase contains a sebacoyl chloride and the aqueous phase contains hexanediamine.

The organic phase may be suspended in the aqueous phase in any suitable way, but according to the invention it is preferred that the organic phase is suspended in the aqueous phase by being sprayed into the aqueous phase by means of a nozzle.

Thus, for example, microcapsules of encapsulated isooctane suspended in an aqueous phase may be prepared by spraying, through a nozzle, isooctane having a content of 10% sebacoyl chloride in an aqueous solution containing hexanediamine and an emulsifier, for example, FC-128, which is the potassium salt of fluorinated alkyl carboxylate from 3M Company. For example, per 5 liters of aqueous solution containing 0.1 kg 1,6-hexanediamine and 1.5 g FC-128, there may be introduced, by spraying, 100500 ml isooctane containing 10% sebacoyl chloride. After the addition by spraying, it is suitable to stir, for example, to slowly stir for about I hour, in order to secure that the reaction is completed Thereafter, the resulting suspension may be allowed to stand until the particles have collected in a top phase, and the lower phase consisting of water, diamine and soap may be withdrawn for regeneration. Thereafter, the top phase is suitably washed 3-5 times with deionized water and thereafter 2-3 times with aqueous phosphatebicarbonate buffer which constitutes the continuous phase in the final suspension.

Subsequent to the last separation which may be facilitated by means of mild centrifugation, one has a suspension which is suitable for synthesizing a reference liquid through equilibration with a gas mixture having a known partial pressure of CO2 and a known partial pressure of 02.

When the organic phase which is to be enclosed in micrdcapsules comprises two organic components which are not mutually soluble, the preparation of the microcapsules according to the invention is performed by suspending one organic component in the other through spraying the first-mentioned component into the second component through a nozzle, and thereafter suspending the resulting suspension in the aqueous phase by spraying it into the aqueous phase through a nozzle. In this way, for example, a suspension of microcapsules, each of which contains perfluorotributyl amine suspended in isooctane, may be prepared.

Now, the especially characterizing features of the reference liquid according to the invention, viz. the oxygen buffer system and the optional hemoglobinsimulating coloring agent have been explained. In the following, the more conventional properties of the reference liquid, i.e. its pH buffer properties and its Pco2 buffer properties will be dealt with.

The establishment of suitable pH and Pco2 buffer mixtures pertains to the known art. In principle, one may, in establishing such buffer solutions, utilize the well-known relationship between carbon dioxide dissolved in water and the pH of the solution: [HCO3-] pH=pKAm4+log. (a modified Henderson-Hasselbalch equation) [CO2] wherein pKA is the thermodynamic dissociation exponent of carbonic acid, m is a constant, ,u is the ionic strength, [HCO3-] is the molar concentration of hydrogen carbonate ion, and [CO2] is the molar concentration of carbon dioxide.

It will thus be seen that the pH of a hydrogen carbonate/carbon dioxide solution is defined and known when the hydrogen carbonate and carbon dioxide concentrations are known. Such systems containing known concentrations of hydrogen carbonate ion and carbon dioxide may be established in various manners, e.g. by equilibrating a sodium hydrogen carbonate solution with a CO2-containing gas of known partial pressure of CO2, but it is also possible to start from sodium carbonate and form sodium hydrogen carbonate in situ by "titration" with the carbon dioxide, and likewise, it is possible to establish a solution with known hydrogen carbonate ion concentration and known carbon dioxide concentration by adding an acid, such as HCI, to a hydrogen carbonate solution or a carbonate solution. These various procedures and the corresponding calculations of the parameters of the established systems are dealt with in more detail below.

The partial pressure of carbon dioxide in a liquid depends on the concentration of dissolved carbon dioxide in the liquid and of the solubility of carbon dioxide in the liquid in question, in the following manner: [C02]t=kt Pco2 wherein kt is representative of the solubility of carbon dioxide in the liquid, which solubility is temperature-dependent. A liquid containing a hydrogen carbonate ioncarbon dioxide buffer system (as shown by the above Henderson-Hasselbalch equation) will, hence, show a fixed Pco2 at a fixed pH.

For the purpose of the present invention, where it is desirable that the reference liquid retains, to the degree possible, its declared values for pH and Pco2 during storing and manipulation, it is desirable to take measures to secure, to the degree possible, that gain or loss of small amounts of carbon dioxide (during storing and/or manipulation) results in as little change in pH and Pco2 as possible, and according to the invention, this is suitably obtained by combining the hydrogen carbonate ion-carbon dioxide buffer system with another pH buffer system, according to the invention suitably a phosphate buffer system. Through this, the total buffer effect is increased with respect to the variation of both pH and Pco2 at loss or gain of small amounts of carbon dioxide. Hence, preferred reference liquids according to the invention contain both a phosphate buffer system and a hydrogen carbonate ion-carbon dioxide buffer system. In analogy with the above explanation, the establishment of these systems may be obtained in various manners, e.g. by equilibration of a phosphate buffer system with carbon dioxide, by equilibration of a phosphate/carbonate buffer system with CO2, by addition of an acid, for example HCI, to a phosphate/hydrogen carbonate buffer system, or by addition of such an acid to a phosphate/carbonate buffer system. These various methods for establishing such buffer systems also pertain to the known art, but will, however, be discussed in greater detail below.

As most of the parameters with respect to which the reference liquid according to the invention is to be used for quality control and/or calibration, are temperature-dependent, the quality control and/or calibration should be performed at a specific temperature for which the reference liquid is adapted, and the packaging of the reference liquid should state at which temperature or in which temperature range the declared parameter values apply and/or can be guaranteed with a particular uncertainty. In connection with the below explanation of the preparation of the reference liquid it will be understood that one may either prepare and pack the reference liquid at the same temperature as the one at which it is later to be used, or one may prepare and pack the reference liquid at a temperature different from the use temperature and then, by means of physicalchemical calculations and/or empirical corrections, determine the parameter values declared for the use temperature on the basis of the parameter values applying at the preparation temperature.

As mentioned above, the reference liquid according to the present invention must be enclosed in a gas-tight container, and according to the invention it is desirable that the reference liquid is enclosed substantially free of gas phase in the gas-tight container. The reason for this is that a possible difference between the temperature at which the declared data of the reference liquid apply, and the temperature at which the container with the reference liquid is opened or punctured for introduction of the reference liquid into the equipment to be checked or calibrated, will result in less changes in the parameters measured on the reference liquid at the measuring temperature in the cases where the reference liquid is enclosed in its container substantially free of gas phase, than in the cases where there is a substantial gas phase present together with the reference liquid in the gas-tight container.

In practice, the packing of the reference liquid in the gas-tight container will be performed under scrupulously controlled conditions with respect to temperature and pressure, and it is important that the container is totally filled up with liquid. Suitably, the container is to a certain extent flexible so that there will be no separation of a gas phase (micro bubbles) at varying barometer pressure and temperature during storing, and suitably, the reference liquid filled into the container shows a reduced total gas pressure, in order to secure that at varying temperature conditions during storing, transportation or at the use, e.g. at temperatures between 0 and 50"C, no formation of micro-bubbles due to supersaturation of dissolved gas will take place.

Suitable containers for packing the reference liquid according to the invention are, e.g. ampoules, cannulla-ampoules, tubules (glass cylinders closed at both ends with metallized plastic or rubber stoppers), metal capsules, vials, and preferably tubes with sealed tip or metal foil tubes. The container should be chemically inert to the reference liquid, in order to avoid any changes in the parameters of the reference liquid due to reactions between the components of the reference liquid and the container material. Hence, if the container is of metal, it will be suitable that it is lined with a material which shields the metal from the reference liquid, for example with a plastic foil such as a polyethylene foil.

Hence, a suitable embodiment of the container is for example a continuous metal and plastic foil container which may be of the shape corresponding to the known coffee bags, Tetra Pak milk cartons (Tetra Pak is a Registered Trade Mark), and shampoo pads, or preferably a plastic-laminated metal tube closed at both ends by welding together the plastic foil. Here, it is essential that the materials and design of the container are so selected that the diffusion through the welding seams is so small that loss of O3 and CO2 during storing, e.g. during two years of storing time, will not change the liquid beyond what is acceptable; for most practical utilities, changes of Pco2 and Po2 of at the most about 2%, preferably at the most about 1%, will be acceptable. Furthermore, it is important that the specific material and thickness of the plastic foil are so selected that the amount of CO2 and O2 absorbed in the plastic foil will not influence the measuring results, e.g. presuming that the containers are filled at 370C and opened or punctured at about 20"C.

A plastic laminated metal tube of the above kind may for example comprise an interior layer of for example 50 y of polyethylene or Barex'TM' 210 (PAN C, polyacrylonitrile copolymer) (Lonza), which is used because of its welding properties (in this case, the tube may be closed by periphery welding). If it is desired to be able to heat-sterilize the reference liquid, polypropylene may be used instead of the above-mentioned material. The metal may suitably be aluminum foil which is diffusion tight. A suitable thickness for the aluminum foil is for example 30 ,u, which is greater then the thicknesses in which aluminum foil is usually employed; the purpose of using the greater thickness is to reduce the risk of "pin holes".

Another possibility is to use several thinner aluminum foils laminated together. If desired, a layer of nylon may be incorporated between the polyethylene and the aluminum foil, for example a nylon layer of 15 , for increasing the strength of the packing, and the exterior layer may be nylon in a thickness of suitably about 12 , or polyester in the same thickness.

Naturally, the container should be designed in such a manner that the reference liquid may be introduced in a blood gas measuring equipment anaerobically, i.e. without access for atmospheric air, and the known art comprises various suitable designs and adaptors.

The exact process selected in the preparation of the reference liquid will depend on the identity of the systems involved in the reference liquid and, for example, on the method selected for establishing the pH system, vide the above explanations. When the oxygen buffer capacity-increasing organic substance is a non-water soluble organic substance showing great solubility for oxygen, and the adjustment of the pH-Pco2 system is performed by equilibration of a buffer system with a gas mixture of exactly adjusted and known partial pressure of CO2 and 02, which gas mixture is suitable prepared in a manner known per se using a CO2 gas supply, an O3 gas supply, an N2 gas supply, a premoistener and a pressure regulating unit, whereby, e.g. for the equilibration, in the course of 16 hours, of 200 liters of reference liquid which does not in advance contain CO2, and with an average utilization of 2% of the gas mixture, there is suitably used a gas flow of about 120 liters/minute, and whereby continuous monitoring of the partial pressure of CO2 and O3 in the gas mixture is performed by means of Pco2/Po2 electrodes which may be controlled by means of a reference gas measuring equipment, the thus equilibrated reference liquid is filled anaerobically and substantially free of gas phase into the containers, and optionally, sterilization is performed in a manner known per se, e.g. by radioactive irradiation. If addition of acid is used instead of the equilibration with carbon dioxide for establishing Pco2, such as mentioned above, this addition of acid is suitably performed after the equilibration with 02.

The reference liquids prepared and packed in this manner should be carefully checked by taking out samples at suitable intervals and measuring these samples by means of Pco2/Po2 measuring apparatus, pH equipment, Hb equipment, and total CO2/O2 measuring equipment, the check on total CO2/O2 being performed to secure that the CO2/O2 capacity is in fact present. Officially recognized reference preparations and procedures may be used for the calibration of the measuring equipment in connection with this checking, like in connection with the fine adjustment of the production parameters.

When the reference liquid according to the invention is used for quality control, it is, as indicated above, introduced substantially anaerobically into the blood gas measuring equipment, preferably in the same manner and under the same conditions as the blood samples for which the blood gas measuring equipment is designed, the prescriptions given on the packing of the reference liquid being, naturally, carefully adhered to, and the reference liquid passes into the measuring units comprised by the blood gas measuring equipment, usually measuring electrodes and, with respect to the possible hemoglobin unit, usually a photometer.

The response of the measuring units on the reference liquid is recorded, and if the recorded values deviate to an unacceptable degree from the declared values of the reference liquid, the problems must be analysed in order to ascertain and remedy errors in the blood gas measuring equipment and/or its calibration liquids and/or the procedure for operating the blood gas measuring equipment.

When the reference liquid according to the invention is used for calibrating blood gas measuring equipment, it is introduced anaerobically into the measuring units of the blood gas measuring equipment, whereby the prescriptions given on the packing of the reference liquid are naturally carefully adhered to, and the measuring equipment is adjusted until there is conformity between the values read from the blood gas measuring equipment and the values declared on the reference liquid. In using the principle of the present invention for calibration, the most suitable way will be to calibrate the blood gas measuring equipment with two different reference liquids according to the invention with values differing from each other, this being quite in conformity with known art, and like the abovementioned Radiometer ABLI equipment, the blood gas measuring equipment will be adapted to automatically perform such calibration, in this case using prefabricated and pre-packed reference liquids according to the invention instead of the known art reference liquids which are made ready for use in the apparatus proper.

Now follow firstly specific prescriptions for establishing the pH/Pco2 system, and thereafter working examples illustrating the reference liquid according to the invention. It will be understood that although a very great exactitude in the parameters of the reference liquid may be obtained already on the basis of the calculations stated in the below prescriptions, using the empirical constants stated, the utmost exactitude of the parameters will depend on a fine adjustment performed in the particular production equipment and based upon partly empirical constants characteristic to the equipment, and partly corrections after control measurements against for example officially acknowledged standards.

Prescription 1 In a buffer system consisting of sodium hydrogen carbonate equilibrated to known partial pressure of CO2, the pH can be calculated according to the Henderson-Hasselbalch equation: [HCO3-]a pH=pKA+log.

[CO2]a pKA is the thermodynamic dissociation exponent. to calculate the pH, it is necessary to correct pKA for the effect of ionic strength. This is done according to the Debye-Hiickel limiting law for activity coefficients. The law is used in approximated form: pK;=pK,-0.495 wherein u is the ionic strength of the electrolytes in the solution, and 0.495 is an empirically ascertained constant. In connection with the introduction of molar concentrations of [HCO3-] and [CO2], the Henderson-Hasselbalch equation becomes as follows: pH=pK0.495/+log[HCO2]lOg[CO2] [CO2] is found from the equation: [CO2]=a. Pco2 10-3 a is the modified Bunsen absorption coefficient.

The pH in a 24. 10-3 M solution of sodium hydrogen carbonate equilibrated with a gas mixture with a partial pressure of CO2 of 40 mm Hg will be 7.53. If indifferent salt (e.g. NaCI) is added to a total ionic strength of 0.21, the pH will be 7.38.

The following constants, valid at 370C, are used: pKA=6.33 a=0.032.

The prescription applies at 370C.

The pH in a 12 10-3 M solution of sodium hydrogen carbonate equilibrated with a gas mixture with a partial pressure of CO2 of 80 mm Hg will be 6.95.

If indifferent salt (e.g. NaCI) is added to a total ionic strength of 0.11, the pH will be 6.84.

The same constants as above are used.

Prescription 2.

When a phosphate buffer is equilibrated with CO2, the pH of the buffer is changed in dependence of the amount of CO2. In a phosphate-hydrogen carbonatemixture system, the following equation will establish itself: HCO3-+H2PO4-=HPO4--+Co2+H2O If the initial concentrations of HCO2-, H2PO46 and HPO4--, respectively, are designated: A, B, C the result, after mixing, is: (A-a)+(B-a)AC+a)+a wherein a is the change which will bring the system into equilibrium. When carbon dioxide is added until the pressure corresponding to the concentration m, another change results: (A-α)+(B-α)=(C+α)+m The mass action equation for the hydrogen carbonate system is: [H+](A-a) =K, m and for the phosphate system: [H+](C+a) K2 (B-α) Elimination of a from the equations results in: ([H+] A)-(K1 m) (K2. B)-([H+] C) [H+] [H+]+K2 This results in a second degree polynomium with respect to [H+], and the pH can be calculated:

D = 4 (C + A) K1 K K2 # # m K, is the first acid strength constant of carbonic acid K2 is the second acid strength constant of phosphoric acid A is molar concentration of hydrogen carbonate B is molar concentration of dihydrogen phosphate C is molar concentration of monohydrogen phosphate.

In the calculations, the following constants are used: pK1=6.328-0.495# (valid at 37 C) pK2=7.029-0.495# (valid at 37 C) In a phosphate buffer wherein C=B, =0.1, and the partial pressure of CO2 is 40 mm Hg, pH will be 6.59. C=B=0.025 mol/liter.

In a phosphate buffer wherein C=4B, and ,u=0.17, and the partial pressure of CO2 is 80 mm Hg, the pH will be 6.90. C=0.0523 and B=0.0131 mol/liter.

In both solutions, the initial concentration of hydrogen carbonate=0: Prescription 3.

By adding hydrogen carbonate to the phosphate buffer solutions according to prescription 2, pH can be varied.

A buffer consisting of C=B=0.022 M, and A=0.012 M, equilibrated with a gas mixture with a partial pressure of CO2 of 80 mm Hg, has pH 6.84 (y=0.1).

A buffer consisting of C=0.047 M, B=0.012 M, and A =0.022 M, equilibrated with a gas mixture having a partial pressure of CO2 of 40 mm Hg, has pH 7.36 (u=0. 17).

Prescription 4.

The sodium hydrogen carbonate in the above prescriptions may be formed by titration of sodium carbonate with carbon dioxide.

In this case, CO3--+ H+HCO3- and CO2+H2OH++HCO3 the sum is: CO2+H2O+CO3--=2HCQ At pH 7, the equilibrium is displaced completely to the right, as less than 0.1 of the carbonate is unconverted.

Hence, in prescription 3, the hydrogen carbonate may be replaced with carbonate in half of the concentration to result in the same pH as calculated in prescription 3.

Prescription 5.

Prescription 1 may be calculated with carbonate instead of hydrogen carbonate. Carbonate is to be used in half of the concentration, cf. the reaction equilibrium in prescription 4.

Prescription 6.

Hydrogen carbonate buffer+acid, e.g. HCI [HCO3-]+[CO2]=Total concentration of "CO2,, when the hydrogen carbonate buffer is not prepared by equilibration with carbon dioxide, it may, for example, be prepared by addition of hydrogen ion from, e.g., hydrochloric acid, to a hydrogen carbonate solution.

Hence, in prescription 1 80 mm Hg of Pco2 corresponds to 2.56x10-3 mol of CO2 per liter which may be formed from hydrogen carbonate ion by addition of acid HCO3-+H+=CO2+H2O To form 2.56x 10-3 mol, 2.56x 10-3 mol of H+ are required, for example in the form of HCI.

This means that (12+2.56)x 10-3=14.56x10-3 mol of hydrogen carbonate to which are added 2.56x 10-3 mol of HCI and sodium chloride to a total ionic strength of 0.11 gives, in 1 liter of solution, a pH of 6.84.

When it is desired to prepare the liquid on the basis of carbonate, the following applies: CO3--+H+=HCO3 HCO3-+H+=CO2+H2O CO3- -+2H+=CO2+H2O 1 liter of solution containing (12+2.56)x10-3 mol of carbonate=14.56x10-3 mol, to which are added (14.56+2.56)x 10-3 mol=17.12x 10-3 mol of hydrogen ion in the form of hydrochloric acid, and sodium chloride to a total ionic strength of 0. I I, also results in pH 6.84.

Prescription 7.

When the buffers mentioned in prescription 3 are prepared by addition of acid in a closed container, a buffer containing C=B=0.022 and A=14.56x10-3 mol/liter admixed with 2.56x10-3 HCI perliter has pH 6.84, and the ionic strength in this solution becomes 0.1025.

When the buffer is prepared from sodium carbonate, the same amount of sodium carbonate is added, while the amount of acid is increased to (14.56+2.56)x10-3=17.12x10-3 mol of HCI per liter.

A buffer consisting of C=B=0.022 and sodium carbonate=14.56x 10-3 mol/liter admixed with 17.12x 10-3 mol of HC1 per liter will show a pH of 6.83, as the ionic strength in this solution is increased to 0.117.

Example 1 A bicarbonate-containing phosphate buffer having a disperse phase of nyloncoated microparticles of silicone oil.

The composition is: In the water phase: disodium hydrogen phosphate 0.047 molal potassium dihydrogen phosphate 0.012 molal sodium hydrogen carbonate 0.022 molal Ponceau 4 R 1.7 g/liter The water phase constitutes 60% of the liquid, the remaining 40% being fluorohydrocarbon (perfluorotributyl amine) emulsified in the water phase.

The organic phase: microcapsules of silicone oil coated with nylon.

Preparation as follows: 1.6 g sebacoyl chloride is dissolved in 80 ml of dimethyl siloxan polymer 200 fluid/100 from Dow Corning. 200 is the type, 100 is the viscosity in centistokes. 35 ml of the mixture is, at a pressure of about 110 atm., sprayed, through a nozzle, into 400 ml of aqueous solution containing 3 g NaOH, 4 g 1,6-hexanediamine (Fluka) and 15 g Tween3 80 (surface active substance from Merck Schuchardt). The mixture is sprayed into the solution under the water surface. The aqueous solution is vigorously stirred during the spraying and for 4 minutes thereafter. On the surface of the silicone oil particles, sebacoyl chloride reacts with 1,6hexanediamine in the aqueous phase with formation of a nylon membrane.

The resulting particles are separated by centrifugation and washed several times with the aqueous phase which is introduced as the continuous phase. The resulting reference liquid contains 40% of aqueous phase and 60% of disperse phase and is suitable for synthesizing a reference liquid having pH=7.36, Pco2=40 mm Hg, Po2=70 mm Hg, and Hb=14 g%, at 370 C, which is done by equilibrating the liquid with a gas mixture having a partial pressure of CO2 of 40 mm Hg and a partial pressure of O2 of 70 mm Hg at a temperature of 37"C.

Example 2 A bicarbonate-containing phosphate buffer with a disperse phase of porous, air-containing silicone rubber.

The composition is: In the water phase: disodium hydrogen phosphate 0.047 molal potassium dihydrogen phosphate 0.012 molal sodium hydrogen carbonate 0.021 molal Ponceau 4 R 1.7 giliter The water phase constitutes 60% of the liquid, the remaining 40% being finely divided porous silicon rubber particles (CAF4/60 "Rhodorsil"), prepared by applying superatmospheric pressure during the curing and subsequently, still during the curing, releasing the pressure.

This liquid is suitable for synthesizing a reference liquid (having a pH of 7.36, a Pco2 of 40 mm Hg, a Po2 of 70 mm Hg, and a Hb of 14 g%, all at 37"C), which is performed by equilibrating the liquid with a gas mixture having a partial pressure of CO2 of 40 mm Hg and a partial pressure of O2 of 70 mm Hg at 37"C.

Claims (17)

WHAT WE CLAIM IS:

1. A synthetic reference liquid for quality control and/or calibration of blood gas measuring equipment, said reference liquid showing, at a fixed temperature, a known pH, Pco2, and Po2 value, said reference liquid comprising an aqueous phase containing at least one pH buffer system and particles suspended in the said aqueous phase, the particles being either 1) polymer-coated microcapsules containing at least one organic substance which shows a greater ability to reversibly contain oxygen than does water, or 2) porous particles of a solid substance.

2. A reference liquid as claimed in claim 1, in which the aqueous phase contains both a hydrogen carbonate ion-carbon dioxide-pH buffer system and an additional pH buffer system.

3. A reference liquid as claimed in claim 2, in which the additional pH buffer system is a phosphate buffer system.

4. A reference liquid as claimed in any of the preceding claims, in which the microcapsules contain a hydrocarbon, a fluorocarbon compound, an ester and/or a silicone oil.

5. A reference liquid as claimed in claim 4, in which the microcapsules contain isooctane.

6. A reference liquid as claimed in claim 4, in which the microcapsuled contain perfluorotributyl amine.

7. A reference liquid as claimed in claim 4 in which the microcapsules contain a mixture of isooctane and perfluorotributyl amine.

8. A reference liquid as claimed in claim 7 in which the ratio, by volume, between isooctane and perfluorotributyl amine in the microcapsules is 7-8:3-2.

9. A reference liquid as claimed in claim 8 in which the volume ratio is about 3:1.

10. A reference liquid according to any of the preceding claims in which the polymer coating of the microcapsules is nylon.

11. A reference liquid as claimed in any of the preceding claims, comprising a coloring component showing maximum or significant absorption at about 500 nm.

12. A reference liquid according to any of the preceding claims, which is enclosed in a gas-tight container substantially free of gas phase.

13. A reference liquid as claimed in any of the preceding claims, in which the suspended microcapsules constitute at the most 80% of the total volume.

14. A reference liquid as claimed in claim 13, in which the suspended microcapsules constitute from 40 to 70% of the total volume.

15. A reference liquid according to any of claims 1--4, comprising particles of porous silicone rubber.

16. The invention according to claim 1, substantially as described.

17. The invention substantially as described with reference to the Examples.