METHOD FOR DETERMINATION OF TOTAL CARBONATE CONTENT ESPECIALLY IN BIOLOGICAL FLUIDS

Abstract: Electrochemical method for determining the total carbonate content especially in biological fluids. According to the invention, using an ion-selective electrode, the concentration of certain ions dissociable from carbon acid or affecting the dissociation balance is first measured for the fluid. The fluid is then mixed with another fluid containing a reactive component that changes the ionic concentration just measured, whereupon the said concentration is again measured by means of an ion-selective electrode, thus producing two different measurement results, from which the total carbonate content can be determined. The measurement preferably relates to the pH of the fluid, while metallic ions such as Cu<2+>, forming a complex with hydrocarbonate ions, are used as the component to be added to alter the pH.

Description: METHOD POR DETERMINATION OF TOTAL CARBONATE CONTENT ESPECIALLY IN BIOLOGICAL FLUIDS The present invention concerns a method for determining the to carbonate content especially in biological fluids, using an i selective electrode for the measurement. The biological fluids referred to are, in particular, human bl and urine, which are analyezed to determine their carbonate cont for diagnostic purposes. The methods currently used for electrochemical determination of carbonate content in fluids are based on two fundamental solutio One of these uses an electrode designed for sensing carbon diox (CO2), while the other uses an electrode sensitive to carbon ions. The first solution is represented by US patent 490 23*1, wh proposes a method for determination of the total carbonate cont of a fluid involving measurement of the amount of carbon dioxide the fluid by means of an element consisting of a renewa electrolyte disposed between a gas-permeable membrane and an io selective electrode. In this method, the sample fluid is premi with an acid to release carbon dioxide. The gassed sample is th pumped toward the said element, while at the same time t electrolyte is also circulated by pumping. When the sample reach the element, the pumping is stopped. The gas then diffuses throu the membrane into the electrolyte solution, and the change occurri in the latter is measured by the ion-selective electrode a translated into a total carbonate content value. The disadvantag of this method are the need for a two-channel fluid pumping syst and the use of a gas-permeable membrane, which may be blocked wi time by constituents in the sample, such as proteins. The second solution is represented by US patent ft 196 056, whic involves the use of an ion-selective carbonate electrode. A described in the patent publication, the sample is premixed wi pM buffer solution containing a mercury/EDTA complexone. This ha the main effect of producing carbonate ions, which are then sense by the said electrode. The drawbacks of this method are related i the first place to the imperfections of the ion-selective electrode such as instability of the signal, long response time, shor durability and sensitivity to other ions and substances. The object of the present invention is to provide a new method fo determining the total carbonate content of a fluid in a simple, fas and accurate manner without the above-mentioned disadvantages o existing methods. The invention is characterized in that the flui is first subjected to a measurement, using an ion-selectiv electrode, of the concentration of certain ions dissociable fro carbon acid or affecting the dissociation balance, that the fluid i then mixed with another fluid containing a reactive component tha changes the concentration of said ions thus measured, and that afte the mixing the said ionic concentration is again measured by mean of an ion-selective electrode, thus producing two differen Measurement .results., rom-whic the .total carbonate content .c n b determined. The method of the invention allows concentration measurement without generation of gases. The advantage of this is that n accurate pressure control and no dialysis membrane is necessary This obviates the danger of'memb a * lockages . which present ..th worst problems with the existing methods. Further, an essential feature in the method of the invention is tha the determination of the carbonate content is based on the chemica l ws '.governing the balance..of diffe e ions In . fluid, i.e. th method has an exact scientific foundation ensuring reliable results An embodiment of the method of the invention is characterized that it uses a fluid containing ions of a metal, e.g. Cu 2+ , Pb Cd 2+ , n 2+ or Zn 2+ , which form a complex with carbonate hydrocarbonate ions. Copper ions are particularly suitable, si reagents containing copper ions are stable and inexpensive. A cop ion reacts with a hydrocarbonate ion as follows: Cu 2+ + 2 HCO3 " > CUCO3 + H2CO3 A similar reaction also occurs with the other metallic io mentioned above. Thus in all cases the reaction involves ions on and is very fast. Another embodiment of the method of the invention is characteriz in that the electrochemical concentration measurement relates hydrogen ions, in other words, that the pH of the fluid containi carbonate is measured. The measurement can be performed using glass electrode, which, unlike the electrodes normally used f testing biological fluids, is fast, accurate and free interference in operation. In an advantageous embodiment of the method of the invention, t carbonate content is determined by using a device consisting of t cascade-connected measuring cells, the fluid containing carbona being stopped in the first cell, where the concentration of giv ions, such as hydrogen ions, is measured with an ion-selecti electrode incorporated in the cell, from where the fluid is th pumped into the second cell, where it is stopped and the said ion concentration is again measured with an ion-selective electro incorporated in the cell, the fluid being mixed between the t cells with another fluid containing a component, e.g. metallic io forming a complex with carbonate or hydrocarbonate ions, alteri the said ionic concentration. The necessary fluid motion can achieved with a single one-channel peristaltic pump, and in gener the required equipment is simple in construction. - n - The second fluid to be added to the fluid under carbonat measurement preferably contains metallic ions such as copper, lead, cadmium, manganese or zinc ions in the form of a dissolved salt, e.g. a nitrate with a concentration in the range of 1X10~3M 2.5xlO" 2 M, said ions forming a complex with the carrier electrolyte, • e.g. potassium nitrate, which may have a concentration in the rang of 0.15M-1M, and a substance inhibiting secondary reactions of th metallic ions, e.g. hydrogen peroxide or Fe3 + ions, preferably in concentration of 1x10"^ - Ixl0 -1 M. In the following, the Invention is described in greater detail b the aid of an example, reference being made to the drawing attached, showing a diagram of set of equipment designed for applying th method of the invention. The equipment shown in the drawing comprises a measuring cell 1, which is provided with an ion-selective electrode ISEl and reference electrode REF1. the potential difference between thes being measured with potentiometer.mVl, a s. econd measuring cell 2, which is provided with an ion-selective electrode ISE2 and reference electrode REF2, the potential difference between thes being measured with a potentiometer mV2, and tubes H , 5, 6, connected to a pump 3 and serving to pass the fluid sample throug the measuring cells. The tube 5 between the two cells is provide with a valve 7 communicating with a. branch * tube Λ through which second fluid can be added to the fluid flowing in the tube 5 betwee the cells. The potential differences measured in the two cells ar transmitted to a computer or a similar calculating device via th potentiometer output conductors, identified with reference number in the drawing. In the most advatageous application of the invention, the flui sample, e.g. a blood sample, for which the total carbonate conten is to _be found otf , is passed -via:the tubes ' ft. 5. 6/through -th measuring cells 1 and 2, and a potentio etric pH measurement i performed in both cells, using glass electrodes sensitive t hydrogen ions. For each measurement, the sample is stopped in t cell. Between the cells, a second fluid, introduced via the tube is added to the sample. This second fluid is preferably a reagen e.g. a mild copper nitrate solution, containing divalent copp ions, potassium nitrate as a carrier electrolyte and hydrog peroxide to inhibit secondary reactions of the copper ions. T copper ions react with hydrocarbonate ions contained in the samp in the manner expressed by the equation on page 3, thus consumi hydrocarbonate ions and producing carbon acid, thereby altering t pH of the sample. Therefore, the pH measurement in the second cell yields a result different from that obtained in the first cell and the total carbonate content of the sample can be calculated fr these two results. The total carbonate content CL is calculated from the formula: where is a constant determined by the size of the sample and t quantity and concentration of the reagent employed, and where 'II [HCO-j [H + ] The subscripts "1" and "2" in the formulas above refer to the p measurements in cells 1 and 2 respectively. The constants Q and are calculated beforehand from the formula (I) and using two buff solutions with a known C_ value. Once the constants have bee determined, the formula can be used to calculate the total carbonat content C_. it is obvious to a-person^_^l___-«_i-J_ the art that the embodiment o the invention are not restricted to the example described above, bu that they may instead be varied in the scope of the claims presente below. Thus it is possible that instead of copper ions, other ion forming a complex with carbonate or hydrocarbonate ions are used e.g. Pb 2+ , Cd 2+ , Mn 2+ or Zn 2+ ions, or that instead of the pH, th concentratio ,of hydrocarbonate-sor carbonat ion is^measured in th cells. Further, it is possible to interchange the sample containin carbonate and the reagent, such as the mentioned reagent containin copper ions, so that the reagent is first brought into the measurin cell 1, where its copper content is measured with a suitable copper selectiv velecitrode * ? whereupon? the-3_B-_β_pie^oπt i i added to the reagent via the tube 8 and the copper content of th mixture thus obtained is measured in the second cell 2 with copper-selective electrode. The total carbonate content of th .sample can^then^be calculated from he^___easure-__-_n*^results obtained

Claims: CLAIMS 1. Method for determining the total carbonate content especially biological fluids, using an ion-selective electrode for measurement, characterized in that the concentration of certain i dissociable from carbon acid or affecting the dissociation bala is first measured for the fluid by means of an ion-select electrode, that the fluid is then mixed with another fl containing a reactive component that changes the concentration said ions just measured, and that after the mixing the said io concentration is again measured by means of an ion-select electrode, thus producing two different measurement results, f which the total carbonate content can be determined. 2. Method according to claim 1, characterized in that it uses fluid containing metallic ions, e.g. Cu 2 " 1* , Pb 2+ , Cd 2 *, Mn 2+ or Zn which form a complex with carbonate or hydrocarbonate ions. 3. Method according to claim 1 or 2, characterized in that the pH the fluid containing carbonate is measured. . Method according to one of the claims above, characterized that the carbonate content is determined by using a devi comprising two cascade-connected measuring cells, whereby the flu containing carbonate is stopped in the first cell, where t concentration of given ions, such as hydrogen ions, is measured wi an ion-selective electrode incorporated in the cell, from where t fluid is then pumped into the second cell, where it is stopped a the said ionic concentration is again measured with an lon-selecti electrode incorporated in the cell, the fluid being mixed betwe the two cells with another fluid containing a component, e. metallic ions forming a complex with carbonate or hydrocarbona ions, altering the said ionic concentration. 5. Method according to claim 2 or 4, characterized in that the sai second fluid contains metallic ions such as copper, lead, cadmium manganese or zinc ions in the form of a dissolved salt, e.g. nitrate, which form a complex with the carrier electrolyte, e.g potassium nitrate, and a substance, e.g. hydrogen peroxide o Fe3 + ions, inhibiting secondary reactions of said metallic ions.

Inventors: IVASKA ARI, LEWENSTAM ANDRZEJ, WAENNINEN ERKKI, IVASKA, ARI, LEWENSTAM, ANDRZEJ, WAENNINEN, ERKKI
Applicants: KONE OY
Application year: 1987
Main classification: G01N27
IPC codes: G01N27