KR830000736B1 - Method for measuring the freezing point lowering - Google Patents

Abstract

The freezing point reduction measuring instrument is used in clinical tests on a specimen liquid. The liq. is under cooled so as to measure the osmotic pressure of the fluid which can be a plasma, tears or dialised liquids such as urine. The instrument consists of a cell(1) in a glass tube which is reduced to the under cooling temp by the insertion of a metallic block(4) in cascaded thermal modules(2,3). For the production of cryohydrate cores in a section of the liq. the temp. is reduced even further. The temp is measured in the condition of coexistence between solid and liq. phases.

Description

Freezing point drop measurement method

1 is a longitudinal sectional view of a freezing point measuring unit using a procell.

2, 4, and 5 are schematic diagrams showing configurations of different devices, respectively.

3 is a time chart illustrating the operation of each part of the apparatus of FIG.

6 and 7 are longitudinal cross-sectional views of the freezing point measuring unit using the discreet cell, respectively.

The present invention freezes the supercooled sample liquid in the case of measuring the osmotic pressure of a fluid sample, such as a plasma sap fluid or a dialysis fluid or urine, which is the subject of clinical examination. It relates to an improved method of measuring the freezing point drop which causes a part of the sample liquid to be overcooled as a stimulus.

About 60% of the living body is occupied by water, and the water exists in the living body in a state in which various substances are dissolved as plasma, cell fluid, and other body fluids. And since the cell membrane of the living body is a semipermeable membrane, the effect of the osmotic pressure of the body fluid generated thereon is very large.

Recently, a device for accurately measuring the osmotic pressure of various body fluids and urine based on the freezing point drop method has been supplied to the market one by one, and the osmotic pressure measurement such as plasma, urine, and sap has been adopted in clinical tests, and dehydration and diabetes insipidus (尿)崩 症) It is helpful for the diagnostic management of hyponatremia, hypersodiumemia, uremia, non-ketone hyperosmolar coma, and postoperative patients.

Therefore, in order to use this effectively, what is required for these osmotic pressure measuring methods and measuring devices is that the microorganisms of samples for measuring precious fluids or leaks, etc. can be measured quickly and artificially in case of emergency or in a short time. It is possible to make continuous measurement necessary in the case of the dialysis solution at the time of dialysis.

However, although there are many osmotic pressure measuring instruments currently on the market, none of them can satisfy the above requirements, in particular, continuous measurement is impossible, and further includes various problems described below. .

The osmotic pressure of the original solution is defined as the pressure generated by the diffusion of pure water into a solution partitioned by a semipermeable membrane, and in the case of dilute solutions, it is directly proportional to the other properties of the solution: freezing point drop, boiling point, vapor pressure drop and the number of moles of solutes contained in the same solution. It is directly measured by using a semipermeable membrane, and indirectly obtained by converting the value of any of the other three characteristics.

However, in the case of body fluids, proteins in body fluids cause thermal coagulation in the boiling point method, and in the case of volatile components such as alcohol in the vapor pressure drop method, a large error occurs. In addition, in the direct method, an appropriate semipermeable membrane cannot be obtained. Since it takes time, only the freezing method is used.

Here, the freezing point drop refers to a phenomenon in which the condensation point of a solution as described above is lower than the condensation point of a pure solvent in proportion to the concentration of water in the solute contained in the solution, and is widely used for molecular weight measurement, but osmotic pressure is also included in the solution. Since it is proportional to the number of molecules (atoms) to be obtained, it becomes possible to obtain | require by measuring the freezing point drop degree as shown by Formula (1).

Where ΔTf is the freezing point drop, R is the gas constant, and Tf is the freezing point (absolute temperature) of the solvent. I ₀ latent heat of fusion of 1 g of silver solvent, C is the number of moles of solute dissolved in 1 kg of solvent, and K _f is the dew point drop.

When the solvent is water, such as a body fluid, the freezing point drop of the solution of 1 mol / kg of water concentration is 1.858 ° C (kf = 1.858 ° C), and the freezing point is -1.858 ° C. On the contrary, if the freezing point of the solution is -1.858 ° C, the water concentration of the solution becomes 1 mol / kg. Thus, osmol is used for the unit of osmotic pressure, and osmotic pressure of the solution of 1mol / kg of water concentration is expressed as 1Osm / kg, but the change of osmotic pressure in body fluid is very small, so 1mOsm / kg which is 1/1000 of 1Osm / kg This is used.

Any osmotic pressure measuring device currently used, for example, as shown in U.S. Patent No. 3,203,226, is a rod-shaped thermometer for detecting a cooling bath and freezing point temperature in which the sample liquid is subcooled several degrees below its freezing point. ) And a head consisting of an electronic vibrating stirring rod for freezing, a measuring circuit, a control part, a display part, and the like. Therefore, in addition to the test tube (discrete cell) containing the sample solution, insert the mister and the stirring rod, and immerse the test tube in the cooling bath to make the test solution supercooled. When the temperature of the sample liquid reaches -5 to -6 ° C, the stirring rod is vibrated rapidly to generate ice crystal nuclei to freeze the entire sample liquid. At this time, the temperature of the sample liquid rises to the freezing point temperature by the release of the latent heat of condensation, and then gradually falls after maintaining a flat state for a while. The temperature of the flat portion, that is, the temperature of the sample liquid in the solid liquid coexistence state is measured with a thermistor, and the freezing point drop is converted into osmotic pressure and displayed.

As described above, the present apparatus and method provide the osmotic pressure accurately in a relatively short time by using the demister, which makes the freezing point clear by providing the freezing stimulus caused by the vibration after freezing the sample liquid and freezing it by vibrating. In terms of being able to measure and using a stirring rod for freezing stimulation, it has the following problems.

1) The stirring rod for freezing and the demister for freezing point measurement must be inserted at the same time in the sample liquid, and it is difficult to micronize the sample because a relatively large test tube is required. This requires a certain space even in the miniaturization of the stirring rod and thermistor, and their miniaturization is limited for the following reasons.

In other words, the freezing vibration strength of the stirring rod is quite strong, and sometimes the outside air is drawn into the sample liquid. The vibration time is short as about 1 second, but it vibrates during the freezing process. Therefore, the force is applied to the thermistor by the vibration of the solid liquid coexistence of the sample, so that the miniaturization in the strength with the thermistor and the stirring rod is limited.

2) In order to reduce the size of the test tube and reduce the amount of sample, it is difficult to reduce the size of the test tube.In order to increase the cooling capacity, a large sized compressor for cooling air and water is used for efficient heat dissipation. The device is also required, and the entire device is enlarged.

3) In the apparatus equipped with the cooling tank using the coolant liquid, gently stir the sample liquid during cooling to make the sample quickly supercooled, to uniform the temperature of the sample liquid, to lengthen the flat part after freezing, and to easily measure the freezing point. The process and apparatus are complicated, such that the test tube is placed in the coolant liquid and rapidly cooled to an appropriate temperature, and then the test tube is pulled out of the coolant liquid and held in the cool air stream and gradually cooled to cool the sample evenly.

4) Because the stirring rod is used as the freezing means, it is difficult to use procells, and therefore, it is difficult to continuously measure or measure a large number of samples in a short time. Any current apparatus requires a dedicated test tube for each sample, and when a device for continuous measurement of the same sample is configured, a quantitative divided injection must be performed each time into the test tube, and a special divided injection device and a transfer mechanism of the test tube are required. The whole apparatus has the drawback that it is mechanically complicated and large.

The present invention solves all the drawbacks of the conventional apparatus and satisfies all the requirements of the osmotic pressure measuring method and measuring apparatus described above, and at the same time, enables automatic and digital display that can eliminate individual differences in the measurement. It is to provide a method having excellent characteristics.

In other words, the method of the present invention spontaneously generates ice crystal nuclei in this part by cooling a part of the sample liquid to a lower temperature (supercooled state) than the supercooling temperature instead of the freezing method by the mechanical vibration of the conventional stirring rod. The main point is to induce freezing of the entire liquid.

In this way, the microfluidization of the sample liquid is realized by inserting only a temperature detector (thermometer) for measuring the equilibrium temperature at the time of freezing, which is the original purpose, without interposing a mechanical vibrating member such as a stirring rod in the sample liquid. In addition, in order not to give strong vibration to the sample liquid, the particles in the sample liquid are less destroyed and can be used as it is for measurement of other yields.

It is a matter of course that the present invention, which is characterized by such a freezing method, can reduce the size of the measuring vessel (test tube) even when the present invention is applied to a conventional disc lead method. (Fig. 6). In addition, in the present application, the sample container is intermittently introduced into the measuring container using a prosel method to measure the device, and thus the device has a small number of moving parts, thereby making it possible to mechanically remarkably simplify the apparatus (FIGS. 1 and 2). .

Further, in the method of using the coolant liquid as the cooling means, replenishment of the coolant liquid periodically. In case of transporting the device, it is difficult to take out the coolant liquid. However, in the present application, the test tube is directly connected by a thermomodule or the like without using the coolant liquid as the cooling means. The above difficulty was overcome by cooling. Furthermore, it is clear that the present invention, which is characterized by freezing means, can also be applied to an apparatus in which a coolant liquid is used as the cooling means (Fig. 7).

Hereinafter, the present invention will be described in detail with reference to the embodiments shown in the drawings.

1 is a longitudinal cross-sectional view of the freezing point measuring unit, and a procell (1) made of a suitable material such as a glass tube is used for contacting the gaskets of the dummies (2) and (3) based on the well-known Perchier effect. It is comprised even if it cools to the supercooling temperature via the metal procell block 4 by this. This series of cooling devices is referred to as the main cooling device (A). The lower part of the procell 1 is provided with a tubular section 5, which is arranged at a lower temperature by the thermos 7 via the tubular metal blank 6 made of metal. It is cooled in the state of overcooling. This cooling device is referred to as local cooling device (B).

Ice cores are formed in the tubular portion 5, but the structure of the overcooled portion of the cell body 1 is not limited to the tubular structure. However, in order to improve the measurement accuracy, it is preferable that the temperature of the overcooling portion does not affect the measurement portion. The procell block 4 and the tubular block 6 are made of metal having good thermal conductivity or in order to further improve the cooling, such as forming a plurality of cylindrical shapes (3) (7) or arranging each of them in an annular manner. Also good.

The procell 1 is configured so that the demister 9 for sample temperature detection can be inserted so that the temperature change of the sample liquid 9 can be detected.

Next, the measurement operation of the device of this embodiment will be described in detail with reference to FIGS.

After the power is turned on, the cooling plate 10 is controlled at a low temperature by an external cooling device C composed of a thermo module 2, a power supply device 14, a current control circuit 5, and a demister 16. The mother stone 3 and the thermo module 7 are installed to cool efficiently. In the drawing, reference numeral 11 denotes a thermally conductive lubricant interposed between the procells 1 and the procell block 4, and the reference numeral 12 denotes a heat insulating material surrounding the freezing point measuring unit, and the reference numeral 13 denotes a thermo module. It is a radiator in contact with the heat radiating surface of (2).

Subsequently, when the start art switch 17 is turned on, the sunpla 19 is operated by the instruction of the microcomputer 18 so that the nozzle 20 is the sample container 21. ), The roller pump 22 is rotated at the same time (the third pump roller ON), and the sample liquid 9 is introduced into the procell 1 via the introduction pipe 24. do.

When the liquid level of the sample liquid 9 is detected by the portcouper 25 and the liquid level detection circuit 26, the roller pump 22 is stopped by the instruction of the microcomputer 18, thereby introducing the sample liquid. It is completed (state of process a "sample liquid introduction" of FIG. 3 operation | movement).

On the other hand, the nozzle 20 is raised after the sample liquid necessary for filling the procell 1 is sucked up, and the air layer is sent to the introduction pipe 24 (FIG. 3 nozzle OUT). The second reference numeral 23 is a pump drive device.

When the introduction of the sample liquid is completed, the transfer device 31 is operated by the instruction of the microcomputer 18 and the demister 8 is inserted into the sheet liquid 9. At the same time, the main cooling device A constituted by the thermo module 3, the power supply device 27, the current control circuit 28, the polarity changer 29, and the thermistor 30 is operated to produce a procell. (1) is cooled and the temperature of the sample liquid 9 starts to fall. (The state of the process b "supercooling" of FIG. 3 operation.)

In this case, by appropriately controlling the current flowing in the thermo module 3 by the proportional control method, it is possible to obtain the effect of uniform cooling like the rapid cooling slow cooling performed when the conventional coolant liquid is used.

In addition, when the flow of the sample liquid is stabilized in the state in which the thermistor 8 is inserted, the transfer device 31 may be omitted by inserting the thermistor 8 into the procell 1. The sample temperature is monitored by the thermistor 8, the resistance voltage conversion circuit 32, the A / D converter 33, and the microcomputer 18, and is further stored in the microcomputer 18 in advance. When the operating temperature T ₁ (FIG. 3 curve S) of the mother module 7 is reached, a local cooling device B consisting of the power supply device 34, the current control circuit 35, the polarity switch 36, and the thermo module. Is activated to start cooling of the procell customs 5 (FIG. 3 curve F). On the other hand, the temperature of the sample liquid 9 gradually reaches the subcooling temperature T ₂ set in advance by the current control circuit 28 of the demister 30.

When the temperature of the tubular part 5 drops (FIG. 3 curve F) and -15-25 degrees C is spontaneous, it freezes in this part, and this is called an ice kernel, and the whole sample rapidly freezes (FIG. 3 operation). Process C state of "freezing point nucleation"). The temperature at which natural freezing takes place varies depending on the concentration of the sample solution, etc., but because it is locally cooled, the influence on the measured value can be ignored.

At the same time as the ice, the thermo module 7 is fed by the polarity switch 36 and the thermo module 3 by the polarity switch 29 to the heating motor HM from the cooling motor CM, respectively. Each thermo module 3 and 7 has a current optimum for the measurement programmed in advance in the microcomputer 18 and the heating amount is controlled.

The sample temperature rises by the latent heat release accompanying freezing, and once becomes a flat portion, but gradually rises to become an inverted S-shaped temperature change (process d "freezing, measuring, thawing" of FIG. 3 operation).

The freezing point temperature is measured as the equilibrium temperature of the solid liquid coexistence state with the minimum point T ₃ of the rate of change obtained by differentiating this inverse S-curve by the microcomputer 18, and then applying the osmotic pressure value by applying the above equation (1). Is calculated and output to the display 37 or the recorder 38.

When the sample liquid is thawed gradually and the sample temperature reaches T ₄ , it is determined that all of the samples have been thawed, and the roller pump 22 is rotated (FIG. 3 roller pump ON) to discharge the sample liquid (process of FIG. 3 operation). e "discharge").

The sample extraction device composed of the discharge nozzle 39, the discharge pipe 40 and the roller pump 22 assists with discharge, and is not necessarily required, and may be directly overflowed from the prosel 1. In addition, the discharge pipe 40 may be used as the introduction pipe 24. Reference numeral 41 in the figure denotes a discharge liquid container.

In this embodiment, each block (4) (6) is heated after freezing because it is necessary to return to the liquid phase when the sample liquid is discharged because procells are used in the test tube. For this reason, a cooling module is used as a heat source, and a thermo module in which the heating is easily performed by switching currents is used. However, the cooling rate can be easily adjusted by controlling the current of the thermo module, so that the sample liquid can be controlled according to the program of the microcomputer 18. It is as mentioned above that uniform cooling of (9) becomes possible.

4 (a) shows an example in which a multi-sample measuring device is assembled, and the sample container 21. … The well-known sample transfer apparatus 43 which can move the parallel to the measurement part main body 42 is comprised. In the drawing, reference numeral 45 denotes the sunplain 47, and the suction nozzle 20 of the sunplain 45 represents the sample liquid 9,. … And washing liquid 47 are alternately sucked. Reference numeral 48 is a standard solution. 4 (b) shows the sample container 21. … The other sample feeder 44 which rotates and supplies is shown, and the code | symbol 46 is a sun fly.

5 is another embodiment of the continuous measuring device. The nozzle 20 is inserted into the sample fluid 49 continuously flowing, and the sample fluid is sucked intermittently by a predetermined amount, so that the osmotic pressure is reduced. To measure.

After the measurement, the sample liquid is discharged from the procell 1, and then the cleaning liquid 47 is suction-washed through the nozzle 51 by the switching valve 50 to enter the next sample. Reference numeral 52 denotes a nozzle for a standard liquid and may be continuously recorded by the car art recorder 53 instead of the frequency display device 37.

Each of the examples described above uses a procell in a test tube (measuring vessel), but it goes without saying that the freezing means of the present invention can also be used in the case of a normal test tube type discide cell. Even in this case, the stirring rod is not required, so the cell can be miniaturized and the sample liquid can be micronized.

FIG. 6 shows the discrete cell 1 'as the discrete cell block 4' of the main cooling device A and the capillary block 6 'of the extreme cooling device B as in the case of the above-described procells. It is enclosed and cooled by the thermo module (2), (3), (7), and the cell (1 ') uses what was thinly formed so that the front end (5') was easy to freeze. However, unlike the case of procells, it is not necessary to defrost after freezing, and therefore, each thermo module 3 or 77 may not be switched to a heating motor. However, the cooling temperature is easily adjusted by the blocks 4 ', 6', and the thermostat modules 3, 7, so that the rapid cooling and slow cooling as in the case of using a conventional refrigerant liquid There is no need to move the cell body up and down each time, and the apparatus and the process are simplified. Moreover, the use of the conventional coolant liquid is not prohibited in the present invention, and a small amount of heat medium is used between the disc lead cell 1 'and the block 4' and 6 'of FIG. 6 or between the block and the thermo module. Instead of the cell block 4 'of FIG. 6, the coolant liquid 4 "enclosed in the coolant liquid container 54 may be used as shown in FIG. 7. However, the latter (FIG. 7) simplifies the apparatus process. You can't expect that much.

In addition, the method and apparatus of the present invention are not limited to osmotic pressure measurement, but also applicable to physical properties obtained by measuring the freezing point drop, for example, the molecular weight measurement. In this case, the microcomputer needs to be programmed to calculate the molecular weight as the freezing point drop instead of the osmotic value.

In the method of measuring the freezing point drop by subcooling as described above of the present invention, a part of the sample liquid is cooled as a freezing means to a temperature lower than the subcooling temperature, thereby naturally freezing the entire portion by the freezing nucleus. It does not require a stirring rod, thereby miniaturizing the cell, and thus minimizing the sample and damaging the sample, thus enabling reuse, simplifying the cooling system, and simplifying the measurement process.

Furthermore, the great advantage of the method of the present invention is that it enables the use of procells, which enables to continuously measure osmotic pressure such as body fluids such as plasma, dialysis fluid, urine, or a plurality of samples in a short time. It is in its power.

The device of the present invention implements the above-described method, and with the use of a microcomputer, it is possible to accurately measure a small amount of sample without opening eyes, and at the same time by using a thermo module and a metal block as a cooling means, it is possible to obtain a sample without complicated devices or processes. The uniform cooling allows the continuous measurement of samples and the rapid measurement of multiple samples, and the small amount of samples and the need for agitation means, simplifying the cooling and measuring devices. As a result, the whole device can be miniaturized and there are many outstanding advantages such as fewer failures.

Claims (1)

In the method for measuring the freezing point drop of the sample liquid by subcooling, a portion of the sample liquid in the subcooled state is cooled to a temperature lower than the subcooling temperature, thereby forming ice crystal nuclei in the portion, and using the ice crystal cores as freezing stimuli. By continuously measuring the temperature of the sample liquid in the solid liquid coexistence state by the latent heat of condensation emitted during the freezing of the whole liquid, the temperature of the minimum point of the change rate was calculated as the freezing point temperature, and immediately after the freezing, the siro liquid was heated. A freezing point drop measuring method characterized by thawing a sample liquid in a solid liquid coexistence state.

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