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Convert Your Freeze Drier to Controlled Nucleation for Less than £10.

Over recent years there has been a growing interest in controlled nucleation and especially in top down nucleation to encourage vertically aligned ice crystals, which on sublimation become vertically aligned pores to improve sublimation rates and shorten freeze drying cycle times. The problem is, of course, that this adds to the purchase price and is not available on many small lab scale freeze driers. Even where retro-fitting is possible, it is not an inexpensive option. Pharmaceutical freeze drying, however, is not the only branch of science which has an interest in controlling ice crystal morphology for subsequent freeze drying.

The ceramics industry uses freeze casting, or ice crystal templating, to create ceramics with aligned, or designed porosity (for example ref 1). In this field, freeze drying is used to remove the template after the ceramic has been cast. Unlike the pharmaceutical industry, however, the ceramics industry has the opportunity to use other solvents for freeze drying, for example camphene (2).

Any reader familiar with the history of tofu will draw our attention to the value of the old literature. Specifically to Kim M.K. and Lugay J.C.who published an article entitled Freeze alignment: A novel method for protein texturization; almost 40 years ago. Using nothing more complex than a pan of soy milk sitting on a block of dry ice, Kim and Lugay grew unidirectional ice crystals, substantially perpendicular to the bottom of the pan, which they then removed by freeze drying.

Controlled nucleation? Well although the time and temperature of nucleation are not controlled, the method leads to bottom up nucleation, and in that sense it is controlled nucleation. There is a significant difference to top down nucleation and that is in the ice crystal growth phase of freezing. To explore this in more detail we need to look at the fabrication of scaffolds for tissue engineering.

The control of pore size, and distribution is critical in the development of biocompatible materials for use as tissue repair scaffolds. Both the ceramics industry and the biocompatible materials scientists have investigated a range of parameters for controlling pore size and distribution, including cooling rates and temperature gradients. Davidenko et al (4) show some interesting results using moulds to create temperature gradients, and one mould in particular, their mould 3, showed rather good vertical alignment of ice crystals. This mould was nothing more sophisticated than a perspex block, with holes drilled into it, sitting on top of a conductive material. What it did was eliminate the radiation and gas phase conduction (collision) elements of heat transfer, creating a uniaxial, linear temperature gradient. A temperature gradient which then guides the vertical alignment of ice crystals. This too is bottom up nucleation, and like the Kim and Lugay experiment has an important difference to pharmaceutical top down nucleation, when it comes to ice crystal growth.

A solution sitting in a vial on a cooled shelf will show a temperature gradient from hot (top) to cold (bottom). In top down nucleation ice crystal growth is therefore hot (faster growth) to cold (slower growth). With the bottom up techniques the reverse is true, ice crystals grow from colder (slower) to warmer (faster) regions.. The superior results of Davidenko et al may be explained by the almost complete elimination of the radiation and gas phase conduction modes of heat transfer, giving more precise control of the temperature gradient.

This suggests a rather inexpensive and simple method of converting any freeze drier to (bottom up) controlled nucleation and growth. First. take a sheet of non-conductive material, and a polystyrene sheet is a readily available, inexpensive alternative to perspex. Next, drill holes all the way through the sheet, creating channels. Sit the sheet on the freeze drier shelf and insert you vials into the holes, so that the vial bottom is in contact with the shelf but the vial wall is shielded from radiation and gas phase conduction. Cool your shelf!

The channels in the polystyrene/perspex mask will need to be a couple of millimetres wider than your vial, as the mask will need to be removed before beginning primary drying – which would be very slow indeed without the radiation and gas phase conduction components of heat transfer.

It isn't a perfect solution. No doubt you will displace some stoppers when removing the mask and the need to open the chamber door to remove the mask is not compatible with the manufacture of sterile material, but, given the price of polystyrene sheeting, you have just converted your freeze drier to controlled nucleation, and still have change from £10. If you save several hours in primary drying, would you begrudge 5 minutes replacing stoppers? A more sophisticated mask might use strips of clear plastic sheeting, available from hobby shops, to line the channels in the polystyrene and reduce friction. It may double the cost, but you will still have spent less than £20!

References

  1. J.M. Rodriguez-Parra, R. Moreno and M.I. Nieto. J. Serb. Chem Soc 77, 1775-1785 (2012).

  2. E-J. Lee, Y-H. Koh, B-H Yoon, H-E Kim and H-W Kim. Materials Letters 61, 2270-2273 (2007)

  3. .Kim M.K. and Lugay J.C. Freeze alignment: A novel method for protein texturization. In: Utilization of Protein Sources. D.W. Stanley, E. D. Murray, D.H. Lees (eds.), Food and Nutrition Press Inc., Westport, Conn., 177-187, (1981).

  4. N. Davidenko, T. Gibb, C. Schuster, S.M. Best, J.J. Campbell and R.E Cameron. Acta Biomaterialia 8 667-676 (2012).

 

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Tony Auffret also writes a regular blog article for the BioUpdate Foundation.

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