Vitech Scientific - Vital Clinical Photometers
If one of these colligative propertieschanges, all the rest change by a predictable amount. In aqueous solutions the thing that can bring about change to these colligative properties is the amount of dissolved material present. In fact the change is directly proportional to the total number of molecules and ions present, irrespective of their size and molecular weight, a large protein molecule will have the same effect as a single Na+ ion.
So one mole (the molecular weight in grams) of any substance dissolved in one kilo of water will cause an osmotic pressure of 17,000 mmHg, a Boiling Point Elevation of 0.52oC, a Vapour Pressure decrease of 0.3 mmHg and a Freezing Point Depression of –1.86oC, although it’s more usual to express this as an Osmolality of 1000 mOsmols per kilo of Water, 1000 mOsmols for short.
Some people confuse Osmolality with Osmolarity. A solution with an Osmolarity of 1000 would contain one mole of substance in one liter of Water, which will clearly be a stronger solution as it won’t contain quite as much water as the one made up with one kilo of water. For example, one mole of sugar made up to one liter will contain less water than a mole of salt made up to a liter due to the extra space taken up by the sugar molecules.
Although both solutions will have an Osmolarity of 1000 the sugar solution will have a much higher Osmolality. It’s usual to refer to Osmolality rather than Osmolarity when dealing with solutions containing mixtures of substances since comparisons can then be made between solutions of different strength irrespective of the molecular weights of the substances dissolved in them
What is the best way of measuring this? Osmotic Pressure, the pressure generated across a semi-permiable membrane with water on one side and the solution you are measuring on the other, would seem to offer the best choice with such high pressures being produced from relatively low solution strengths. In practice it is impossible to measure due to the problem of getting a perfect semi-permiable membrane, small ions will always get through.
Even if the perfect membrane did exist the pressures exerted are so immense, 23 atmospheres for 1000mOsmols, that it would be sure to burst! Of course, if you want know about only the larger molecules, such as proteins, you can measure Osmotic Pressure with a Colloid Osmometer and select an appropriate membrane with a cut-off of 10,000 or 20,000 Daltons which will hold back the protein molecules but allow everything else to get through. But this is no use if you need to know the total Osmolality.
Measuring Boiling Point Elevation is messy and the change in BP is only small, clearly this is not an option for blood samples, unless you want a blood curdling experience, so we are left with Vapour Pressure and Freezing Point Depression. Vapour Pressure works, but there is a drawback when measuring solutions containing other liquids which exert a vapour pressure of their own, for example a blood sample containing alcohol will give a misleadingly high result.
So we come to Freezing Point Depression, which doesn’t have any of the drawbacks associated with the other methods. You need to be able to measure temperate very accurately, 1 mOsmol will produce a temperature change of only 0.0018oC, but it is a robust method and the one in universal use in Clinical Chemistry Labs and Quality Control Labs throughout the World.
In practice the sample is supercooled to a predetermined temperature, lower than the expected freezing point, as shown on the Standard Freezing Curve. Freezing is initiated either by a physical shock or in some cases by seeding with a small crystal of salt or a very cold stir wire. This produces a water-ice mixture which remains at the freezing point plateau for long enough to measure the temperature, provided you have an accurate thermometer, usually a calibrated thermistor, selected to give a linear response. Freezing Point Osmometers are calibrated at two points against accurately made up salt solutions with a known freezing point and then checked with a control solution with a known value around 290 mOsmols such as Clinitrol.
What are the main applications? Well pretty well every Hospital Biochemistry Lab in the World will have one for measuring blood and urine osmolality. The body does a good job of maintaining blood at a constant osmolality of around 290 mOsmols so any departure from this can be significant. It won’t tell you what’s wrong with a patient, but taken together with other observations or tests it can assist diagnosis or enable a course of treatment to be monitored.
A raised osmolality can often occur after surgery or with severe burns patients due to evaporation and an Osmometer can not only be used to detect this but also monitor the infusion of liquids to correct it. The Kidneys concentrate urine and Renal Function can be measured by comparing serum and urine levels. A 24 water deprivation test requires hourly osmolality measurements of both. Because they are so quick and easy to use Osmometers can often be found in Intensive Care Units and Special Burns Centres for immediate use as well as in the Biochemistry Lab.
The application for quickly screening patients admitted into Casualty in a coma, for alcohol poisoning has already been mentioned. In this case a significant difference between measured and calculated osmolality, derived from measuring electrolytes and adding a bit more for glucose and urea, can point to the likelihood of alcohol being present. This is known as the “the Osmolar Gap”, although it should more correctly be referred to as “the Osmolal Gap”!
But Clinical applications aren’t the only use to which an Osmometer can be put. Recently Sports Physiologists have been using them with elite Sportsmen and women for measuring early morning urine samples as an indicator of hydration levels. Anything over 600 mOsmols indicates that more water should be drunk. If the reading is over 1000 mOsmols it is regarded as a warning not to take part in any strenuous exercise until the body has been re-hydrated. It’s actually quite dangerous to exercise whilst de-hydrated.
Another area where Osmometers are proving useful is Quality Control Labs, especially those producing solutions used for injection or cell culture, like Pharmacies and IVF Clinics, or for eye treatment, such as eye drops and contact lens wash solution, or for checking that so-called “isotonic Sports Drinks” are in fact iso tonic. In fact osmolality is often used to ensure that formulations have been made up correctly as this is usually much quicker and cheaper to do than a full chemical analysis.
Osmolality is also used to monitor the progress of fermentation processes when large molecules are broken down into smaller ones with a corresponding increase in osmolality. One advantage of Osmometers in all the above applications is that it only takes a minute to get a result and no sample preparation is necessary.
Osmometers come in all shapes and sizes. If there is not a lot of sample available, which may be the case for clinical applications, then micro Osmometers are available which work with samples as small as 20µl. If there is plenty of sample available, which is usually the case with the QC applications, then other models are available which work with larger samples of 200µl.
In summary, Osmometry is a useful analytical tool, often overlooked, because it quickly measures everything in the sample rather than a specific constituent, but vitally important if the overall solution strength is critical.