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The maximum fi ll volume should be to set to the maximum safe storage volume for the tubes minus the % accuracy and the % precision of the liquid handling system being used.
Using the example below for the 0.7ml External Thread tube, the calculated safe working volume is 525ul. This generates a maximum internal pressure of 2Bar, however due to the large sample volume compared to the low headspace volume, only a small error in the target fi ll volume can produce a signifi cant increase in pressure. A fi ll error of just 5% will result in an internal pressure of over 3Bar and will likely lead to the tube cracking or the push cap popping off.
Figure 5: Calculating ‘headspace’
Figure 9: Sample at 20C v Sample at freezing temperature Controlled rate freezing Figure 6: Calculating safe working / storage volumes
Another important consideration is the method of freezing. Ideally the sample should be frozen in a controlled rate method, to ensure that the sample is frozen from the bottom upwards allowing it to expand when freezing. If the sample freezes too rapidly, it can become super cooled before nucleation occurs and the phase change from liquid to solid (ice) takes place.
Figure 7: Impact of Charles Law on Sample Volume
As can be seen by the graph in Figure 7, as you get closer to the maximum safe working volume, the pressure inside the sample storage tube increases exponentially.
This is due to the ratio of sample volume to headspace. This highlights the importance of knowing the fi ll volume of the sample tube, which also requires knowledge of both the accuracy and precision of the liquid handling system being used.
If this happens an ice plug can form at the top of the sample, limiting the space available for the rest of the sample to expand as it freezes. As the sample cannot expand vertically due to the ice plug, the resulting force is horizontal causing the polypropylene to crack.
Figure 10: Example of TPE cap popping during thawing
Figure 11: Ice plug forming at the top of the sample, leading to tube damage Figure 8: Fill error of 5% leads to the tube cracking
Azenta Life Sciences offers a range of products that enable controlled rate freezing. Products are identified in the following posters: ‘Best Practices for introducing -80°C Bio-Samples into -190°C Vapour Phase Liquid Nitrogen Freezers - with Experimental Evidence of Thermal Excursions’ and ‘Temperature Controlled Manipulation and Alcohol-free Cryopreservation: A new era in Sample Handling and Biobanking’.
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