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Today we'll introduce in detail the instrument used to measure osmolality—the osmometer.
Applications ✦
Osmolometers are widely used in production and daily life. In pharmaceuticals, drug analysis, and clinical medication, osmolometers can determine the osmolality of intravenous infusions, nutritional solutions, electrolytes, or osmotic diuretics; in medical diagnosis and emergency care, they are used to measure the osmolality of body fluids, helping doctors quickly diagnose patients' conditions; in criminal investigations,osmolometersare used to detect drugs, aiding police in quickly solving cases; in environmental monitoring, they can measure the osmotic pressure of plant leaves; in measuring cell culture media, they can determine whether cells are isotonic; …
Surprisingly, osmometers have such wide applications that even commonly used eye drops require osmometer testing!
Determination Method ✦
The osmotic pressure of a solution is mainly determined using four methods: vapor pressure reduction, boiling point elevation, freezing point depression, and osmotic pressure measurement via a semi-permeable membrane. In practice, direct measurement of osmotic pressure is difficult, while freezing point depression is convenient, easy to perform, highly accurate, requires small sample sizes, and has no denaturing effect on biological fluids, making it suitable for various biological fluids and foods. Currently, most osmolality meters available domestically and internationally are designed based on the freezing point depression method, which is widely accepted by clinical and pharmaceutical researchers.
Freezing Point Depression Method
Based on Raoult's freezing point theory, the freezing point depression method is based on the proportional relationship between the freezing point depression and the molar concentration of the solution. It uses a highly sensitive temperature-sensing element to measure the freezing point of different solutions, thereby determining the osmolality of the measured solution.
Experiments show that the freezing point of pure water is 0℃. If 1 osmolality of a solute dissolves in 1 kg of water, the freezing point of the water will decrease from 0℃ to -1.860℃. Therefore, the osmolality of a solute in a solution can be calculated by measuring the freezing point depression. The formula is as follows:
The freezing point is the temperature at which a solution using water as a solvent changes from a liquid to a solid state; it is also the temperature at which the solution reaches its equilibrium state of ice and water coexisting. During the continuous cooling of an aqueous solution, the temperature at which the solution reaches or even falls below its freezing point without freezing is called the supercooling temperature. When a solution reaches its supercooling temperature, it is extremely unstable; introducing ice crystals or disturbances will cause freezing. The process can be seen in the following diagram illustrating freezing:
As shown in the schematic curve of the freezing process, when the test solution cools from room temperature to the set supercooling temperature, the ice-crystal probe is automatically inserted into the test solution, immediately inducing crystallization. During the transformation from liquid to solid, molecular energy shifts from a high-energy state to a low-energy state, and the excess molecular energy is released as heat, known as the "heat of crystallization." This causes the temperature of the test solution to rise briefly and reach an ice-water equilibrium state. The temperature remains relatively constant for a short period, providing an accurate temperature measurement platform for the temperature measurement system. The temperature measured at this time is the freezing point temperature of the test solution.
Recommend Instruments ✦
Theosmoremeterdeveloped by Huatai Hehe (Beijing) Trading Co., Ltd. utilizes the freezing point depression principle and features a stylish and convenient color touchscreen LCD with automatic probe raising and lowering. It employs a dual cooling system, resulting in short pre-cooling time, fast detection speed, and convenient continuous testing. It requires small sample volumes, meeting the testing needs of various fields. It can simultaneously display the osmolality, freezing point, and molar ratio of the sample. It supports two-point and multi-point linear calibration. The cooling system uses a heat-conducting-free design, eliminating frequent maintenance. It includes the names of hundreds of injectable drugs from the Chinese Pharmacopoeia for easy pre-setting of sample data.
Osmoremeters are widely used in production and daily life, and their regular calibration is crucial, directly affecting the accuracy of the instrument's measurements. The "JJG1089-2013 Metrological Verification Procedure," which our company participated in drafting, specifies the initial verification, subsequent verification, and in-use inspection of osmolality measuring instruments using the freezing point depression principle.
The calibration items and metrological performance indicators of a freezing point osmolality meter mainly include three aspects: indication error, repeatability, and stability.
Calibration Items and Metrological Performance Indicators
1. Indication error
The instrument was preheated and pre-cooled according to the instruction manual to complete the calibration. An appropriate amount of the standard substance to be tested was taken using a pipette, and then the nominal osmotic concentrations of the standard substance were measured at 100, 200, 300, 400, 500, 600, and 700 mOsmol/kg. Each measurement was repeated three times, and the arithmetic mean of each measurement was calculated.
When the concentration does not exceed 400 mOsmol/kg, the instrument indication error ∆ξ1+i is calculated according to formula (1); when it exceeds 400 mOsmol/kg, the relative indication error ∆ξ2+i is calculated according to formula (2).
Example
When measuring a standard substance with a nominal concentration of 100 mOsmol/kg,
Unit: mOsmol/kg
| Measured values | 1st time | 2nd time | 3rd time |
101 |
100 |
100.8 |
|
| average value | 100.6 |
||
Substituting into formula (1), we get:
The error result is 0.6 mOsmol/kg, which does not exceed the error requirement of ±6 mOsmol/kg, and therefore complies with the relevant regulations on indication error.
When measuring a standard substance with a nominal concentration of 500 mOsmol/kg, if the standard substance is >400 mOsmol/kg, it should be calculated according to formula (2).
Unit: mOsmol/kg
| Measured values | 1st time | 2nd time | 3rd time |
501 |
502 |
500 |
|
| average value | 501 |
||
Substituting into formula （2）, we get:
The indicated error is 0.2%, which does not exceed the ±1.5% error requirement, therefore it meets the relevant regulations for indicated error.
Measure the standard substances with concentrations of 100, 200, 300, 400, 500, 600, and 700 mOsmol/kg three times each. Substitute the values into the two formulas according to the concentration range to calculate the indicated error. All values must meet the requirements.
2. Repeatability
After preheating the instrument according to the instruction manual and precooling the refrigeration system, measure the standard substance with a nominal osmolality of 300 mOsmol/kg and record the instrument reading. Repeat the measurement six times, and calculate the standard deviation s using the following formula. This is the repeatability of the instrument.
Example
Data were measured 6 times for a standard solution with a nominal concentration of 300 mOsmol/kg.
Unit: mOsmol/kg
| Measured values | 1st time | 2nd time |
3rd time | 4th time | 5th time | 6th time |
299 |
301 |
300 |
300 |
301 |
301 |
|
| average value | 300.3 |
|||||
Substituting into the formula, we get:
The repeatability result is approximately 0.817 mOsmol/kg, which meets the error requirement of ≤2 mOsmol/kg, and therefore complies with the relevant regulations for repeatability results.
3. Stability
After the instrument is preheated according to the instructions and the refrigeration system is precooled, measure the standard substance with a nominal osmotic pressure molar concentration of 300 mOsmol/kg, and record the instrument indication. Then measure every 10 minutes, for a total of 7 measurements. Calculate the difference between the maximum value ξmax and the minimum value ξmin of the 7 measurement results according to the following formula, which is the stability of the instrument within 1 hour;
Example
Measure the values of a standard solution with a nominal concentration of 300 mOsmol/kg seven times.
Unit: mOsmol/kg
| Occurrence | 1st time | 2nd time | 3rd time | 4th time | 5th time | 6th time | 7th time |
| Numerical value | 299 |
301 |
300 |
300 |
300 |
301 |
299 |
The instrument's stability result after seven measurements was 2 mOsmol/kg, meeting the error requirement of ≤2 mOsmol/kg, and therefore complies with relevant stability regulations.
The determination of osmolality is widely used in pharmaceuticals, drug analysis, and clinical drug use; it is also used in medical diagnosis and emergency care to measure the osmolality of body fluids; and it can be widely applied in biological, botanical, environmental, hygiene product, food and beverage, and other fields for the determination of aqueous solution osmolality and scientific research.
Huatai Hehe's osmometers are chosen by various testing fields for their user-friendly design, high measurement accuracy, good reproducibility, and convenience. To make it easier for users to operate the instrument, we have compiled common problems and solutions during use for your reference:
1. Natural Crystallization and its Handling
▪ Natural crystallization refers to the phenomenon where the sample automatically crystallizes during the instrument's supercooling process, even before ice crystals are inserted by the probe.
▪ Automatic crystallization is often caused by unclean test tubes, impurities in the sample, unmelted ice crystals on the temperature probe, or reuse of test tubes.
▪ Natural crystallization will display "Sample naturally crystallized, please replace the sample and retest" on the touch screen.
▪ Methods to remove natural crystallization:
① For solutions with high salt concentrations, dilute the sample to prevent salt crystallization;
② If crystals adhere to the surface of the temperature probe, clean it and wipe it clean with filter paper;
③ Replace the test tube.
2. Causes and Handling of Slow or No Crystallization
When crystallization is slow or non-crystallizing, the touch screen will display "Sample not crystallized, please replace the sample and retest." Data exceeding 3500 mOsmol indicates invalid data (unless crystallization occurs in special cases). ▪ If the sample's osmolality is too high, its freezing point will be very close to the probe insertion point temperature. This will make it difficult for the sample to crystallize after probe insertion (especially noticeable in viscous solutions). In this case, the sample must be diluted.
▪ Probe crystallization unsuccessful:
① The humidity in the probe's environment is too low, or the instrument starts testing very shortly after startup, before ice crystals have formed on the probe. In this case, lift the probe cover to expose the probe briefly.
② If the probe with ice crystals does not insert into the sample when it is moved from a high position downwards, the start motor key on the rear panel should be used to adjust the probe position so that it can accurately contact the supercooled test solution.
③ If the probe encounters obstruction during its downward movement or if water droplets in the probe guide hole cause the ice crystals on the probe to melt, use a special suction bulb to remove the water droplets from the probe guide hole.
3. Factors Causing Large Deviations in Test Data
▪ Is there a significant difference between the molar concentration of the standard solution used for calibration and the actual sample being tested? A standard solution with a molar concentration close to that of the sample should be used for calibration.
▪ Are the standard solution and sample freshly prepared? Experience shows that standard solutions and samples can change after two hours in a dry environment; therefore, please use freshly prepared standard solutions and samples for instrument calibration and testing.
▪ Storage requirements for standard solutions: protect from light, keep cool, and seal tightly.
▪ Are the test tubes replaced for each test? Reusing disposable test tubes can cause errors.
▪ Do the test tube specifications meet the requirements? SMC series instruments have high precision; therefore, differences in test tubes can cause measurement errors. It is recommended to use test tubes recommended by Tianhe Instruments.
▪ Has the pipette tip at the sampler been replaced? The pipette tip should be replaced when testing different samples.
▪ Are the instrument's temperature probe and probe contaminated? The temperature probe and probe should be cleaned with the sample after each sample change.
4. Precautions
▪ When testing different samples consecutively, the temperature probe and probe are easily contaminated. Therefore, when changing samples, inject twice the amount of the sample to be tested or pure water into the test tube. Push the test tube into the support to the stop position, ensuring the temperature probe is completely immersed in the sample within the test tube. Press the start motor button on the rear panel to lower the probe at least three times. Remove the test tube, replace it with a new test tube and sample, and repeat the above steps at least twice (increase the number of operations for samples with large concentration differences) to clean the temperature probe and probe. Finally, use filter paper to absorb any remaining liquid.
▪ A new test tube, standard solution, and sample must be used for each calibration or test.
▪ After the instrument is turned off, if it needs to be turned on again within 20 minutes, be sure to absorb any melted water from the upper cooling tank and probe with filter paper. Otherwise, the probe may freeze, causing instrument malfunction due to human error.
▪ If the ambient humidity is >60% or the testing speed slows down, check and remove condensation caused by excessive air temperature in the cooling tank. Use absorbent paper cut into a column and insert it into the cooling tank to absorb the water.
▪ Do not use organic solvents for testing or cleaning (the instrument is only for testing and cleaning with aqueous solutions).
▪ After testing highly viscous samples, use a cleaning bottle to clean the probe and probes.
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