There are two common adjustments when making buffers or solutions in a research laboratory. The first adjustment is always pH and the second is osmolarity. The reason pH is adjusted first is because osmolarity is more important for biological systems and measured on a finer scale than pH.
Osmolarity is a measure of the amount of free molecules in solution. It’s important to adjust osmolarity when working with biological samples. Both mammalian cells and plant cells have water-permeable membranes due to aquaporin channels and the free diffusion of water. Cells, however, generally don’t pass larger molecules or ions as freely through the cell membrane. Some sodium channels leak but sodium/potassium pumps work nonstop to keep a diffusion gradient across the cell membrane.
Because of differences in salt or free molecule concentrations inside a cell versus outside a cell, osmosis (the attraction of water to other molecules to balance concentration gradients across water-permeable membranes) will cause water to move either into or out of the cell. When working with cells, isotonic (i.e. same osmolarity) buffers should be used in accordance with the natural physiological osmolarity of the biological sample.
Calculating Osmolarity
Osmolarity is measured by the quantity of free molecules in solution. To be able to calculate osmolarity by hand one needs to know how molecules in the proposed buffer act in water. Salts dissociate and therefore each resulting ion should be considered a unit. Molecules like sucrose or ethanol, which don’t dissociate in water, are counted as a single unit.
For each molecule the molecular weight needs to be known, as does the weight added to the solution. From this, calculate the millimoles in solution and multiply it by the number of units a molecule dissociates into when in the solute (usually water). When recording the value for osmolarity it is written as [x]mOsM.
Osmolarity v. Osmolality
Osmolality is the measure of millimoles per kilogram of solute while osmolarity is a measure of millmoles per liter of solution. For making buffers it is more appropriate to measure osmolarity, though some people (erroneously) use the terms interchangeably.
Using an Osmometer
The most common osmometer used on biological research laboratories is a 5520 made by Wescor, which calculates osmolarity by measuring vapor pressure. Operation of the osmometer is very simple, yet very technically taxing. Every action must be repeated in applying samples. The more consistent the movements of the technician, the more accurate the osmometer result will be.
Basic steps:
- Open the chamber door
- Place a small circular filter on the metal sample chamber
- Add 10ul of the buffer to be measured, touching the droplet to the circular filter
- Close the chamber door and wait until the timer in the window shows you the value of the sample
- Record the value
- Clean the sample chamber
To calibrate the osmometer, follow the same steps required to run a sample when running an osmolarity standard but instead of recording the value, press select (which will move an arrow to the word “calibrate”) and press “enter” (which will calibrate the machine). It’s recommended that the osmometer is calibrated before each use.
Regulating the osmolarity in solutions is oftentimes neglected or overlooked, yet it can often make the difference between experimental success and failure and should never be underestimated.
 |
