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Exercise 9: Serum Osmolality

  • Page ID
  • RELATED READINGS: Pages 265-271. See Methods in CD-ROM for Osmolality


    Upon completion of this exercise, appropriate discussion, and related reading, the student will be able to:

    1. Determine serum osmolatlity using a vapor pressure osmometer.
    2. Determine serum osmolality using a freezing point osmometer.
    3. Estimate the osmolality by calculation, given the concentrations of several components of a sample.
    4. Discuss possible explanations for discrepancies seen in the results obtained using the three different methods of testing.


    Osmolality is an expression of the concentration of disolved particles (solute) in a specific amount of solution (solvent). This determination can be made directly or indirectly. The direct methods are usually based on the freezing point or the vapor pressure of a solution. The indirect method involves measuring the concentrations of major solutes and then calculating the osmolality. Each method has certain advantages and shortcomings. The reference range for serum osmolality is in the 280-300 mOsm/kg of water range.


    • Standards
    • Vapor Pressure Osmometer (Wescor)
    • Freezing Point Osmometer (Advanced or Precision Type)
    • Serum Samples
    • Controls


    Students can perform serum CO2 determinations prior to or as part of this exercise. Alternatively, you can provide CO2 values for each of the samples used. This exercise provides an opportunity to illustrate how “other factors” can corroborate an abnormal anion gap. Students will first need to determine that the anion gap is abnormal and then, using a serum chemistry profile or “case history” can evaluate the validity of the abnormal results.


    1. For each control or sample that you were assigned, determine the osmolality using both types of instruments that are available. Refer to the operating instructions dealing with the specific instrument you will be using. Record your results on the data sheet. Make a notation of the appearance of the serum samples you tested.
    2. Your instructor will give you the concentrations of Na, K, BUN, and glucose of one or more of your samples. Using equations 14-2 (p.267) and ones from the Osmometry method in the CD-ROM and calculate the serum osmolalities. Record these values on the data sheet.


      1. Your instructor will provide you with the following solutions: 100 mmol/L NaC1 and 100 mmol/L urea.
      2. Measure the osmolalitity of each solution and record on the data sheet.
    1. Prepare a hemolysate of red blood cells, as described in Exercise #15, Bilirubin. Add a very small volume of this hemolysate; for example 10 L, to 1 mL of a sample whose osmolality had previously been measured. Record on data sheet.
    2. For those laboratories who have access to both freezing point depression and vapor pressure osmometers, this experiment can demonstrate the effect of a volatile solute on osmolarity.
      1. Prepare an ethanol stock solution (75000 \(\mu\)g/mL, 1630 mmol/L) by adding 10 mL of 95% ethanol to 100 mL of distilled water.
      2. Place 1 mL of a serum whose osmolality had previously been measured and calculated into each of 2 small glass test tubes.
      3. To one labeled “control”, add 10 \(\mu\)L of distilled water. To the other labled “spike” slowly add 10 \(\mu\)L of the ethanol stock solution while gently vortex-mixing the sample. Mix both solutions.
      4. Measure the osmolality by both freezing point and vapor pressure osmometers.
      5. Record on data sheet.
      6. Calculate the osmolal gap, as described by Equation 14-3, p. 267 of Kaplan and Pesce.

    NAME: ___________

    DATE: ___________


    Appearance Vapor
    Osmolarity, mmol/L
    Normal Control
    Abnormal Control
    Sample #
    Sample #


    A. Osmolality, mmol/L
    0.1 M NaC1 ________________
    0.1 M urea ________________

    B. The hemolyzed sample was (circle one) mildly, moderately, grossly hemolyzed.

    Previously measured osmolality (sample #____) = ____________mmol/L
    Hemolyzed sample’s osmolality = ____________mmol/L
    C. Vapor Pressure Freezing Point
    Osmolality, mmol/L
    Unspiked Sample
    Ethanol spiked sample
    Osmolality gap


    1. $$\text{calculated mOsm/L} = 2 \cdot \text{Na (mmol/L)} + \frac{glucose\; mg/L}{180\; mg/mol} + \frac{BUN\; mg/L}{28\; mg/mmol}$$
    2. $$\text{calculated mOsm/L} = 1.86 \cdot \text{Na (mmol/L)} + \frac{glucose\; mg/L}{180\; mg/mol} + \frac{BUN\; mg/L}{28\; mg/mmol}$$


    1. Does hemolysis appear to interfere with either instrumental method?
    2. Provide a plausible explanation of any discrepancy seen in the results, using the different methods.
    3. What appears to be the major advantage/disadvantage of each method?
    4. How does the presence of a volatile solute affect the three methods of determining osmolality? What is the effect of a volatile solute on the osmolality gap?
    5. Can you explain any difference in the osmolalities of the 0.1 M NaC1 and 0.1 M urea solutions?