Sample Integrity Focus How safe are your samples?


Part II – Working Volume Alexis MacLeod, Azenta Life Sciences, alexis.macleod@azenta.com


Plastic storage tubes are used in laboratories worldwide to store biological and chemical samples across a wide range of temperatures from ambient to -196°C. Knowledge of the working volume is a key criterion to consider when selecting the most appropriate tube and, is dependent on a range of factors including:


• Fill volume of the tube • Accuracy of the volume dispensed • Freezing conditions • Thawing conditions • Cap selected


• Burst pressure of the cap


Manufacturers of sample storage tubes specify the size of tubes in a variety of ways, but rarely state the sample type or storage conditions required to achieve the volumes stated. This can lead to catastrophic results if the working volume of the tube is exceeded.


Manufacturers use nomenclature such as: • Tube size • Nominal volume • Working volume • Safe storage volume


Figure 1: Nominal volume vs working volume. Fill volume


The impact of overfi lling is more profound for biological samples with a high water content, which, due to its unique properties, creates challenges when storing and freezing samples.


When water freezes, the molecules form a crystalline structure maintained by hydrogen bonds which push the molecules further apart. As a result, solidifi ed water,


i.e. ice, is less dense than the liquid format, which is why ice cubes fl oat in our drinks.


Figure 2: Water molecules vs water molecules in solid ice in liquid water.


A consequence of this is that the volume occupied by water increases by approximately 9% as it freezers. It is this expansion of the volume that can lead to cracking of sample storage tubes if the working volume is exceeded, with a resultant loss of precious samples.


As we can see from Figure 3, the lowest density and therefore the largest expansion of water (and by association biological samples), occurs at the freezing point (0°C for pure water and slightly lower for biologicals).


The impact of Charles Law on sample volume


Charles’ Law states that the volume of a fi xed mass of gas is directly proportional to the temperature at a constant pressure. As the temperature of the sample decreases, the air fi lling the space between the sample and the cap (headspace) will also decrease at a linear rate at temperatures between 20°C to -80°C.


Even if the extent of the ‘Over Fill’ is not enough to crack the tube, the expansion of the sample elevates the pressure in the tube which can lead to the cap or O ring ‘popping out’ (Figure 4). What happens when an internally threaded tube is overfi lled by only 5% - increased pressure in the tube causes the ‘O’ ring to pop out with loss of tube integrity.


Figure 3: The lowest density and therefore the largest expansion of water occurs at the freezing point.


To address this issue, Azenta Life Sciences exploited the co-moulding technique to weld an integrated TPE-gasket to the polypropylene cap, in place of a traditional silicone ‘O’ ring, eliminating the potential loss of sample integrity associated with expulsion of the O ring.


Azenta Life Sciences was also the fi rst to develop the External Thread 96 format sample storage tube, which has the benefi t of maximising the internal volume and therefore working volume for a given height of tube. Unlike the traditional internal thread tube, additional sample volume can be accommodated as the cap does not ingress into the tube.


Calculating the safe working volume


The safe working / storage volume of a tube can be calculated to ensure the internal pressure generated during freezing does not exceed the burst pressure of the sealing method.


2Bar pressure should be considered the maximum safe pressure even if the seal is capable of a higher pressure in order not to damage the tube itself.


Figure 4: The expansion of the sample in the tube can lead to the cap or O ring ‘popping out’.


LAB ASIA - FEBRUARY 2025


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