Investigating Crystallisation Kinetics with Ultrasonic Spectroscopy

Crystallisation in palm oil

Crystallisation of Lysozyme

Crystallisation in palm oil

The above figures show the ability of ultrasonic spectroscopy to monitor crystallisation. Monitoring of crystal formation is essential for optimisation of process control in the batch crystallisation of various compounds.

In this example high-resolution ultrasonic spectroscopy was used to assess the crystallisation in palm oil subjected to different thermal treatments.  Palm oil is a complex natural oil consisting of two main triglyceride fractions, palmitic and oleic acid as well as small amount of other triglycerides and diglycerides. The temperature was cooled down from 40C to 10C at 1C/min and at 0.2C/min, and  crystallisation under cooling was monitored by measuring both ultrasonic velocity and attenuation.  The above shows two distinctive transitions, one at 35C and the other below 20C. Both transitions are seen clearly in the slow cooling regime. High temperature transition is attributed to crystallisation of palmitic fatty acid, while low temperature point associated with the crystallisation of oleic fatty acid. Rise in ultrasonic velocity is proportional to the growth of solid phase (micro-crystals), and thus it can be used to make quantitative analysis of kinetics of the crystallisation. At 17C, the solid fat content in the oils of the two different cooling rates is the same, as indicated by similar values of ultrasonic velocity.  The crystal network formation is also shown by a clear transition resulting in an increase in ultrasonic attenuation for both cooling rates (Figure (b)). The sharp peak at 35C shows a high cooperativity of crystallisation at low cooling rates.

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Crystallisation of Lysozyme

Lysozyme protects us from bacterial infection by attacking bacterial cell walls, causing them to rupture.  By varying the experiment conditions, various amounts and sizes of protein crystals can be produced.  The amount of crystals as well as an assessment of the size of crystals and interaction between crystals is an important part of routine analysis in the pharmaceutical industry. 

Crystallisation of lysozyme was analysed using the HR-US 102 spectrometer, as shown in the figure above.  In this measurement 1ml of a solution of lysozyme (40mg/ml) in 0.1 M sodium acetate pH 4.8 was placed into the ultrasonic cell.  A precipitating agent was added to the sample and kinetics of crystallisation was followed.  The figure above shows 3 stages in the crystallisation process.  Over the first 3.4 hours of the reaction, no significant change is detected.  At the end of Stage (I) the ultrasonic velocity and attenuation start to increase due to the formation of crystals.  This increase continues through Stage (II) as the concentration and size of the crystals grow.  The rise in ultrasonic velocity, is caused by the increase in rigidity (decrease in compressibility) of the sample as a result of the formation of crystals.   The rise of the ultrasonic attenuation can be attributed to scattering of the ultrasonic wave on the solid crystals formed.  The scattering contribution is dependent on the ratio of crystal size and frequency (e.g. the attenuation at the higher frequencies is more sensitive to the formation of small particles).  Therefore multi-frequency ultrasonic attenuation measurements allow analysis of the crystal structure and size.  During Stage (II) the ultrasonic velocity starts to decrease.  In Stage (III) the ultrasonic attenuation nearly levels off indicating the end of crystal formation in the micron range size.

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