Osmometry is a method of determining the concentration of solute particles in a solution that add to the osmotic pressure. The flow of a solvent (in biological systems, water) through membranes that separate two solutions is regulated by osmotic pressure. The ability to choose molecules of various sizes and shapes varies depending on the pore size of the membrane. Selective membranes enclosing the glomeruli of the kidney and capillary vessels are examples of biologically relevant selective membranes since they are permeable to water and almost all small molecules and ions but not to larger protein molecules.
Differences in the concentrations of osmotically active molecules that cannot cross a membrane allow certain molecules that do cross the membrane to travel in order to achieve osmotic equilibrium. Osmotic pressure is generated by the passage of permeable and solute ions.
Colligative properties
When the solute is introduced to the solvent, it lowers the vapor pressure of the solution below that of the pure solvent, in addition to increasing osmotic pressure. The boiling point of the solution is elevated above that of the pure solvent, while the freezing point is reduced below that of the pure solvent as a result of the difference of vapor pressure. These four properties of solutions—
- Increased osmotic pressure,
- Lowered vapor pressure,
- Increased boiling point, and
- Decreased freezing point
—are called colligative properties.
The colligative effects of solutions are multiplied by the amount of dissociated ions produced per molecule when an electrolyte in solution dissociates into two (in the case of NaCl) or three (in the case of CaCl2). Many solutions, however, do not behave in the perfect case due to incomplete electrolyte dissociation and interactions between solute and solvent molecules, and a 1-molal solution can have a lower osmotic pressure than theoretically predicted. The osmotic activity coefficient is a metric that is used to correct or deviate from the system’s “ideal” behavior:
Osomolality and osmolarity
The total number of solute particles per mass of solvent is directly proportional to the total number of solute particles. The term osmolality refers to concentrations in terms of mass of the solvent (1 osmolal solution equals 1 Osmol/kg H2O), while osmolarity refers to concentrations in terms of volume of solution (1 osmolar solution equals 1 Osmol/L solution). Since solution concentrations expressed in weight are temperature independent, while those expressed in volume differ with temperature, osmolality (Osmol/kg H2O) is a thermodynamically more precise term. The term osmolality is what the clinical laboratory tests, despite the fact that the term osmolarity is often used in medical literature.
Osmolality = Osmol/(Kg H2O) = ϕnC
Where ϕ = osmotic coefficient,
n = number of particles into which each molecule in the solution potentially dissociates
C = molality in mol/kg H2O
Glucose has an osmotic coefficient of 1.00, while sodium chloride has an osmotic coefficient of 0.93 at serum concentrations, leading to 1.86 Na+ (mmol) in the calculation for calculating plasma osmolality (NaCl theoretically adds two osmotically active particles, 0.93 = 1.86). The osmotic coefficient of ethanol is 0.83. The overall osmolality or osmotic pressure of a solution is proportional to the number of all solute organisms’ osmotic pressures or osmolalities. The electrolytes Na+, Cl, and HCO3 contribute the most to serum osmolality when they are found in comparatively high concentrations. Nonelectrolytes including glucose and urea, which are usually present at lower molal concentrations, contribute less, and serum proteins account for less than 0.5 percent of overall serum osmolality.
Determination of Plasma and Urine Osmolality
The measurement of plasma and urine osmolality is helpful in the diagnosis of electrolyte and acid-base imbalances. Na+, Cl-, glucose, and urea are the main osmotic substances in normal plasma; this predicted plasma osmolality is measured using the following analytical equation:
mOsmol/kg = 1.86[Na+(mmol/L)] + glucose [mmol/L]+urea[mmol/L]+9
Other osmotically active molecules in plasma, such as K+, Ca++, and proteins, are represented by the 9 mOsmol/kg added to the previous equation. Notably, 1.86 is two times of osmotic coefficient of Na+, indicating the contributions of both Na+ and Cl–. The + 9 mOsmol/kg parameter is excluded from certain versions of this equation.
mOsmol/kg = 2 x [Na+(mmol/L)] + glucose [mmol/L]+urea[mmol/L]
Plasma osmolality should be between 275 and 300 mOsmol/kg as a reference range. The level of hydration can have a significant impact on the osmolality of the urine. Osmotic concentrations, for instance, can drop to as low as 50 mOsm/kg H2O after excessive fluid intake, but concentrations of up to 1400 mOsm/kg H2O can be seen in people who strictly restrict their fluid intake. Values between 300 and 900 mOsm/kg H2O are generally observed in people who consume an average amount of fluids. After 12 hours of fluid restriction, if a random urine sample has an osmolality of more than 600 mOsm/kg H2O, it is generally safe to infer that the renal concentrating ability is normal.
Measurement of osmolality
Any of the four colligative properties discussed previously—vapor pressure, boiling point, freezing point, and osmotic pressure—could theoretically be used as a basis for osmolality calculation. However, because of its simplicity, freezing point depression is the method of choice in diagnostic laboratories. In addition, unlike vapor pressure, freezing point depression is unaffected by temperature fluctuations.
Freezing Point Depression Osmometer
The components of a freezing point depression Osmometer include the following:
- A thermostatically controlled cooling bath or block maintained at -70 C.
- A rapid stir mechanism to initiate freezing of the sample.
- A thermistor probe connected to a circuit to measure the temperature of the sample.
- A LED display that indicates the time course of the freezing curve and the final result.
The thermistor probe and stirring wire are centered in the sample, which is lowered into the bath and gently stirred to a temperature several degrees below freezing point (-7 0C). The sample is lifted to a point above the liquid in the cooling bath when the LED display signals that proper super-cooling has occurred, and the wire stirrer is adjusted from a gentle rate of stir to a momentary vigorous amplitude, which starts freezing off the super-cooled solution. Just the slush level of freezing occurs, with around 2% to 3% of the solvent solidifying. The emitted heat of fusion warms the solution at first, then the temperature plateaus and stays stationary, suggesting the equilibrium temperature where both freezing and thawing of the solution are taking place.
Vapor Pressure Osmometer
A vapor pressure Osmometer is another kind of Osmometer. However, osmolality calculation with these instruments is specifically related to a decrease in dew point temperature of the pure solvent (water) caused by the decrease in vapor pressure of the solvent by the solutes, rather than a change in vapor pressure (in millimeters of mercury). The inability of the vapor pressure technique to use any volatile solutes found in the serum in its calculation of overall osmolality is a significant clinical distinction between it and the freezing point depression Osmometer. Volatile substances such as ethanol, methanol, and isopropanol escape from the solution and increase the vapor pressure of the solvent (water) rather than lowering it. Because of this, using a vapor pressure osmometer to detect osmolal gaps in acid-base disturbances is impractical, and most clinical laboratories do not recommend using this type of osmometer.
Osmolal gap
An osmolal gap is a measurement of variance within the measured osmolality of a blood sample and the calculated osmolality established on electrolyte concentrations in the same sample. The normal osmolal gap range is usually between -10 and +10 mOsm/kg. Elevated osmolal gaps may suggest the presence of toxins or metabolic disorders such as hyperglycemia or the presence of alcohol or methanol.
Reference:
Tietz Textbook of Clinical Chemistry and Molecular Diagnostics